A quantitative study of alpha-naphthyl acetate esterase-positive cells in non-Hodgkin's lymphomas and reactive lymph nodes.

The numbers of alpha-naphthyl acetate esterase (ANAE)-containing cells (other than T lymphocytes) in non-Hodgkin's lymphomas (NHL) and reactive lymph nodes have been counted, using the Reichert-Jung (Kontron) MOP-AMO3 user-controlled image analyzer. Twenty specimens of NHL and ten reactive nodes were examined. Cells were demonstrated by their content of acid alpha-naphthyl acetate esterase (ANAE) in fixed frozen sections. It was found that lymphomas of high-grade malignancy contained much larger numbers of ANAE-positive cells (10.8-20.5%) than those of low-grade malignancy (1.4-4.1%). The number of ANAE-positive cells (1.4-3.2%) in reactive lymph nodes was similar to that in low-grade NHL nodes.     

Expression of the Acid α-Naphthyl Acetate Esterase Marker by Activated and Secondary T Lymphocytes in Man

Acid alpha-naphthyl acetate esterase (ANAE) activity is characteristic of resting human T lymphocytes. The expression of the ANAE marker by activated human T and B lymphocytes (blasts) and by corresponding 'secondary' lymphocytes has been investigated. Human blood lymphocytes were stimulated by selective T-cell (phytohemagglutinin (PHA) and concanavalin A (Con A)) or B-cell (Staphylococcus aureus strain Cowan 1) mitogens or in the mixed lymphocyte culture (MLC), and the percentage of blasts expressing the marker was quantiated. Whereas 95% of Con-A-activated blasts expressed the marker, approximately 25%-30% of MLC-activated blasts and only 10%-25% of PHA-activated blasts were ANAE-positive. After reversion to secondary lymphocytes, the PHA- and MLC-activated cells regained the ANAE activity, and more than 90% of the blast-derived secondary T lymphocytes were ANAE-positive. Only 2%-8% of blast cells activated by Staphylococcus aureus strain Cowan 1 were ANAE-positive. We therefore conclude that ANAE is not a reliable marker for T cells when activated cells (blasts) are considered.     

Distribution of acid alpha-naphthyl acetate esterase among human T lymphocyte subsets

Human T lymphocyte subsets, identified by means of OKT3, 4 and 8 monoclonal antibodies, were isolated by a fluorescence activated cell sorter (FACS IV) and analyzed for distribution of alpha-naphthyl acetate esterase (ANAE) activity. As compared to OKT8⁺ lymphocytes a higher proportion of OKT4⁺ lymphocytes was ANAE-positive exibiting a spot or dot-like pattern in the cytoplasm. OKT8 and 4 positive subsets showed a similar ANAE distribution in diffuse granular form. Although OKT4 and OKT8 populations presented a different ANAE dot-like reactivity, this marker did not allow as clear a distinction between them as that reported for T_G and T_M lymphocytes.     

Hassall's corpuscles in the thymus of fetuses, infants and children: immunological and histochemical aspects

Hassall's corpuscles (HC) were examined for immunological and histochemical markers in cryostat sections of thymus from fetuses, infants and children. HC could not be detected before 14 weeks of gestation. Receptors for the Fc part of IgG (Fc gamma R) were demonstrated by adherence of ox erythrocytes sensitized with anti-ox IgG using a closed chamber technique. Fc gamma R were also detected by immune complexes of horseradish peroxidase (HRP) and rabbit antibodies to HRP, and with an anti-Fc gamma R serum, using indirect immunofluorescence technique and indirect immunoperoxidase technique. The staining was seen along the outer cell membranes of the HC. The Fc gamma R activity was highest in early fetal life, and decreased with increasing age. Indicator cells which detect receptors for the Fc part of IgM and for the activated third component of complement did not adhere to HC. At 14 weeks of gestation, HC showed a weak alpha-naphthyl acetate esterase (ANAE) activity, while from 16 weeks the staining intensity and pattern was unchanged. In some HC, separate cells with strong ANAE activity were seen. These cells also showed endogenous peroxidase activity, and were stained by an antibody to HLA-DR antigens. Such cells were not seen until 16 weeks of gestation. HC were stained by antibodies to IgG in fetuses older than 16 weeks, and the intensity increased gradually up to 24 weeks. Antibodies to IgM weakly stained some HC in fetuses between 16 and 36 weeks of gestation, whereas antibodies to IgA stained a minority of HC in fetuses older than 24 weeks.     

The ontogeny of acid alpha-naphthyl acetate esterase activity in the thymus

The acid alpha-naphthyl acetate esterase (ANAE) activity was examined in tissue sections of thymus from 24 fetuses, infants and children. ANAE activity was first detected in thymocytes at 11 wk of gestation, and the fraction of stained cells increased gradually until 18 wk of gestation. Thymocytes with circumscribed deposits of ANAE activity were observed as early as at 11 wk, whereas thymocytes with a diffuse, granular staining pattern were not observed before 16 wk of gestation.     

The form factor of alpha-naphthyl acetate esterase-positive cells in non-Hodgkin''s lymphomas and reactive lymph nodes

The shape of alpha-naphthyl acetate esterase (ANAE)-positive cells (other than T lymphocytes) has been measured in 40 lymph nodes. The specimens comprised 15 high-grade lymphomas, 15 low-grade lymphomas and 10 reactive lymph nodes. The parameter used for the measurement of shape was form factor (FF), which is readily calculated by the Reichert-Jung (Kontron) MOP-AMO3 user-controlled image analyzer. Perfectly round cells have an FF value of 1.0, whereas the FF of irregularly-shaped cells diverges from unity. It has been demonstrated that the ANAE-positive cells in high-grade lymphomas have mean values for FF of 0.8-0.9, whereas in low-grade lymphomas and reactive nodes the mean value is 0.4-0.5. Thus, high-grade lymphomas contain many more rounded ANAE-positive macrophage type cells than do low-grade lymphomas and reactive nodes. In the latter two types of specimen there is an excess of branching and spindle-shaped ANAE-positive cells.     

Lymphocyte subsets in human adenoids and tonsils. Rosette formation, alpha-naphthyl acetate esterase cytochemistry, monoclonal antibodies and peanut lectin reactivity

The distribution of mononuclear cell subsets has been studied in human adenoids, tonsils and peripheral blood (PB) by evaluating the presence of surface immunoglobulins, E-rosette formation, receptors for IgG Fc and for complement, alpha-naphthyl acetate esterase (ANAE) cytochemistry, reactivity with peanut lectin (PNA) and with monoclonal antibodies (McAb) (OK panel). Adenoids and tonsils, compared to PB, contain (1) fewer macrophages and T cells but more B cells; (2) higher proportions of ANAE negative, complement receptors and Ia-like antigens bearing T lymphocytes; (3) higher percentages of cells reacting with the McAbs OKT9 and OKT10 ("immature" lymphoid cells). In both adenoids and tonsils, clusters, formed by a central heavily ANAE stained interdigitating cell surrounded by lymphocytes with a sickle-shaped ANAE reaction, were found. Analogous clusters have been previously described in mice and human thymus. Two major hypotheses could be put forward: (1) adenoids and tonsils contain "immature" lymphoid cells undergoing education process, or (2) the above organs contain lymphocytes activated by a constant exposure to bacterial antigens or mitogens.     

Lymphocyte subpopulations in the neonate: high percentage of ANAE+ cells with low avidity for sheep erythrocytes

In the present study we have stained for alfa-naphthyl acetate esterase (ANAE) cord blood lymphocytes (CBL) as well as cord blood E rosetting cells. The results obtained showed that the percentage of E rosettes (E+) is lower in cord blood than in adult peripheral blood when rosetting is carried out with untreated sheep red blood cells (SRBC), while there is no significant difference if SRBC are previously treated with 2-aminoethylisothiouronium bromide (AET). ANAE-positive cells (A+) were higher in cord than in adult blood. ANAE staining of E+ cells showed that CBL include a high percentage of A+ cells with low avidity for SRBC which could represent immature lymphocytes related to the T-cell lineage.     

Disorders of the lymphatic homing and MIF production by the lymphocytes of thymus, spleen and lymph nodes after liver injury caused by portocaval shunt in rats

This paper presents the quantitative changes and disorders in migratory inhibition factor (MIF) production by thymus, lymph node and spleen cells after liver injury provoked by the porto-caval shunt (PCS) in rats. The great depopulation of the lymphatic organs, particularly the thymus, was demonstrated. The reduction of MIF generation after PCS was also noticed. The severe liver injury reflected by high activity of GOT and GPT was observed on the second day after surgical procedure. Our investigations bring into light the multifold interrelationships between the functionally efficient liver and lymphatic organs in rat.     

Stromal cell types in the developing thymus of the normal and nude mouse embryo

The anatomical distribution of various nonlymphoid cell types in the embryonic mouse thymus in vivo and in vitro, as well as in the thymic rudiment of the nude mouse embryo, has been studied. For this purpose a panel of monoclonal antibodies, ER-TR3, 4, 5, 6 and 7, directed to various types of stromal cells of the mouse thymus, was used in combination with immunoperoxidase labeling on frozen sections. It was shown that as early as day 13 in thymic ontogeny distinction of TR4+ cortical epithelial cells and TR5+ medullary epithelial cells is possible. Thus, as far as stromal components are concerned, the thymus at day 13 in ontogeny is already subdivided into cortex and medulla. At day 13, Ia (TR3) was expressed in a focal pattern in the medulla subsequently appearing throughout both cortex and medulla by day 16. The thymic rudiment of the nude mouse embryo differs markedly from the normal embryonic thymus in its lack of demonstrable Ia antigen. Furthermore, TR4 and TR5 were only expressed on occasional epithelial cells lining the cysts of the nude thymus in a mutually exclusive fashion. The majority of stromal cells of the nude thymus, however, is negative for all ER-TR antibodies tested. In addition, we have shown that in organ cultures the organization of the stroma of thymic lobes remains intact, at least for a period of 11 days. Embryonic thymi cultured in the presence of deoxyguanosine, which causes depletion of lymphoid cells, also contain cortical and medullary areas as identified by the presence of TR3,4+ and TR5+ stromal cells. This indicates that the lack of organization in the nude thymus is not simply due to the absence of lymphoid cells.     

Functional rearrangement of lymphohemopoietic system in heterochronically parabiosed mice

This study examines some characteristics of the lymphohaemopoietic system of heterochronically parabiosed CBA mice. It was found that a decline of the primary immune response in a young mouse sutured with an old one is accompanied with the diminution of: bone marrow and thymus cellularity; thymus weight; haemopoietic stem cells (CFC-S) and granulocyte-macrophage precursor cells (CFC-C) contents in the femur, and stromal precursor cell (CFC-F) contents both in the femur and thymus. All the indices tested, including the immune response level, in old parabionts vs. old single mice remained unchanged. In the spleen, the considerable variations of splenic weights in the mice of different control and experimental groups have been found. At the same time, no significant changes in the cellularity and CFC-F number in the spleen of all examined groups of animals have been observed. The findings strongly indicate the considerable rearrangement of lymphohaemopoietic and stromal tissues in the central organs of the immune system, namely, bone marrow and thymus of young mice due to the parabiosis with old ones.     

Classification of normal and malignant lymphatic cells using acid phosphatase and acid esterase

Summary The usefulness of cytochemical tests (APh and ANAE) to replace or to supplement membrane markers in subclassification of normal and malignant lymphatic cells was investigated. Material: normal lymphocytes subfractionated by rosetting and centrifugation, and in M. Hodgkin and CLL; lymphoblastoid cell lines; malignant lymphatic cells in different types of lymphatic leukemias. In normal human blood, T-lymphocytes are marked by a distinct dot-like ANAE-reactivity which is somewhat less pronounced in the small (11%) subgroup of Fc-IgG-receptor positive T-lymphocytes; B-lymphocytes are negative or finely granular positive. Lymphoblastoid cell lines of B- and of T-type are ANAE- and APh-positive. In some lymphatic malignancies, a characteristic pattern of activity of APh or of ANAE may support the diagnosis. The value of ANAE-cytochemistry is highly estimated for the quantitative determination of the percentage of normal T-lymphocytes in lymphatic leukemias, immunological disorders, and during immunosuppressive therapy.

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Abbreviations ANAE acid -naphthyl acetate esterase - APh acid phosphatase - (c-)ALL (common-type) acute lymphatic leukemia - CLL chronic lymphatic leukemia - E(AET)-lymphocytes lymphocytes spontaneously forming rosettes with (AET-treated) SRBC - Fc-IgG-lymphocytes lymphocytes forming rosettes with IgG-sensitized ORBC - MGG May-Grünwald-Giemsa staining of blood films - Non-E-lymphocytes lymphocytes not forming rosettes with SRBC (from interphase of Ficoll-separation) - ORBC ox red blood cells - SRBC sheep red blood cells - (T)-ALL (T-cell-type) acute lymphatic leukemia - T-IgG-lymphocytes E(AET)-lymphocytes forming rosettes with IgG-sensitized ORBC - T-non-IgG-lymphocytes E(AET)-lymphocytes non-rosetting with IgG-sensitized ORBC     

Histochemical identification of T and B areas in paraffin-embedded lymphoid tissues by demonstration of alpha-naphthyl acetate esterase (ANAE) activity.

A strong activity of acid alpha-naphthyl acetate esterase (ANAE) in T lymphocytes and macrophages within mouse lymphoid organs is demonstrable even after paraffin-embedding, if a rapid embedding procedure with acetone as dehydrating agent is used. The lymphoid tissue structure is better preserved in paraffin sections procured after this rapid embedding procedure than in frozen sections commonly used for histochemical demonstration of ANAE. In the paraffin sections the pattern of the ANAE reaction allows a clear delineation of the T and B areas in the lymphoid organs, and the intracellular location of the focal ANAE activity in T lymphocytes is readily discernible.     

Lymphatic system of mice born from mothers treated with ampicillin or cloxacillin during gestation

Ampicillin, an antibiotic, widely used for combating bacterial infections exerts great influence on cells of the immune system of mouse and man. We have studied the effect of ampicillin and cloxacillin treatment of mice in the final week of pregnancy on the development of the lymphatic system of their offsprings. The mice born from antibiotic or saline treated mothers were examined 30 days after delivery. The examination of relative organ weight, cellularity and histopathological picture of lymphatic system (thymus, spleen and lymph nodes) and some other organs was performed. In the group of mice from ampicillin treated mothers we have found decreased relative weight of thymus and spleen and increased weight of lymph nodes with increased cellularity in thymus and lymph nodes. In the group of mice from cloxacillin treated mothers increased cellularity of thymus and lymph nodes was found. The histopathological study of lymphatic organs structure did not reveal any specific changes but the symptoms of focal degeneration and fat necrosis were found in livers of mice born from ampicillin treated mothers. Moreover, in mice born from antibiotic treated mothers the significant lymphocytosis in the peripheral blood was assessed. It was accompanied by an increase of granulocyte number in offsprings from ampicillin treated mothers and increase of monocyte number in those of cloxacillin treated. In conclusion, it could be suggested that ampicillin treatment during pregnancy would exert some effect on the development of lymphopoietic system of fetuses.     

Monoclonal antibody 4C7 recognizes an endothelial basement membrane component that is selectively expressed in capillaries of lymphoid follicles

In order to define compartment-related structures within the extracellular matrix of human lymphoid organs, monoclonal antibodies (MAbs) were generated by immunizing mice with stromal fragments of human tonsils. One MAb (4C7) was selected which recognized an endothelial basal membrane component that is selectively expressed in capillaries of lymphoid follicles. The epitope was also present in follicles within chronically inflamed synovial membrane and in a hyperplastic thymus of a patient with myasthenia gravis. B-cell non-Hodgkin's lymphomas with a follicular growth pattern expressed the antigen in neoplastic follicles, whereas diffuse growing lymphomas lacked the antigen. The restricted distribution pattern suggests involvement of the 4C7-defined antigen in the organization of the follicular compartment within human lymphoid tissue.     

Response of human T lymphocytes to phytohemagglutinin (PHA) after sequential depletion of monocytes, HLA-DR+, Leu11a+, and Leu7+ cells

The experiments presented in this paper deal with the question of whether there is an absolute requirement for alpha-naphtylacetate esterase (ANAE)-positive monocytes, HLA-DR+, Leu11a+, and/or Leu7+ cells to stimulate human peripheral blood T lymphocytes by phytohemagglutinin (PHA). Purified (p) T lymphocytes containing less than 0.1% ANAE-positive monocytes were isolated from human peripheral blood mononuclear cells (MNC) by sequential removal of carbonyl-iron phagocytic cells and of low-density cells by density gradient centrifugation and isolation of E-rosette-forming cells (E-RFC). These pT-cells were further depleted of HLA-DR+, Leu11a+, and/or Leu7+ cells using monoclonal antibodies and cell sorting. The T lymphocytes were stimulated by PHA in an ultra-micro culture in glass capillaries at a volume of 1 microliter or 2 microliters, containing 1000 cells per culture. With this method, the accessory cell requirement could be studied under limiting cell number conditions. The results show that pT-cells can be stimulated by PHA in the absence of ANAE-positive monocytes. No ANAE-positive monocytes were found in the culture after stimulation, indicating the lack of differentiation into ANAE-positive monocytes from ANAE-negative precursors. A rabbit antiserum against leukocytic pyrogen (LP, also containing anti-IL 1 activity) only reduced but did not abrogate the stimulation of pT-lymphocytes by PHA. Addition of adherent cells resulted in an enhancement or in an inhibition of the response of pT-lymphocytes to PHA, depending on cell concentration and culture time: The lower the number of cultured T lymphocytes and the shorter the culture time, the higher was the enhancing activity by additional adherent cells, and vice versa. Further purification of the pT-cells using monoclonal antibodies and cell sorting led to the finding that depletion of either HLA-DR+, Leu11a+, or Leu7+ from pT-cells only reduced but did not abrogate the stimulation of the pT-cells by PHA. However, in absence of HLA-DR+ and Leu7+ cells, the pT-lymphocytes totally failed to respond to PHA. This abrogation of the response was not observed when pT-cells were depleted of HLA-DR+ and Leu11a+ cells. In addition, T11+/HLA-DR- T lymphocytes isolated from E-RFC by positive selection in a cell sorter also responded to PHA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Relationship of Fc-IgG and Fc-IgM receptors to the antigens defined by OKT-Antibodies and the acid α-naphthyl-acetate-esterase spot within human T cells

The expression of receptors for Fc-IgG and Fc-IgM on human T cells was correlated to the antigens recognized by monoclonal antibodies of the OKT series (T3, T4, T8, I1, M1) and to the presence of the dot-like cytoplasmic α-naphthyl-acetate-esterase activity (ANAE). ANAE spots were demonstrated in cyto-centrifuged smears; Fc-receptors (Fc-R) were shown using rosetting techniques which also served to enrich the respective cell populations; OKT antigens were detected by indirect immunofluorescence and OKT4 or OKT8-enriched subsets were obtained by antibody and complement-dependent lysis. Total T and Fc-IgM-R expressing T cells had a similar distribution of OKT antigens (90-95% T3⁺, ～ 60% T4⁺, ～ 30% T8⁺, ? 9 % M1⁺). The Fc-IgG-R expressing T-cell fraction differed in that it contained less OKT3⁺ cells (72 %), about 34 % OKM1⁺ cells and had an elevated percentage of OKT8⁺ cells (53 %). Similarly, OKT8-enriched cell fractions expressed more Fc-IgG-R (44 %) than unseparated T cells (12 %) or OKT4-enriched T-cell populations (16 %). Modulation of the Fc-IgG-R by exposure to immune complexes did not alter the expression of OKT antigens. Fc-IgG-R were, however, not restricted to OKT8⁺ cells, as simultaneous visualization of OKT antigens and Fc-R revealed that 7-11 % of OKT4⁺ cells were also Fc-IgG-R-positive. Thus Fc-IgG-R appear to be preferentially expressed on OKT8⁺ cells, but also OKT4⁺ cells were able to express Fc-IgG-R.As regards the dot-like ANAE activity, T cells expressing Fc-IgM-R exhibited 80-90 % of ANAE positivity, whereas only 20-30% of the Fc-IgG-R-bearing T cells were ANAE⁺. Stimulation of T cells expressing Fc-IgM-R with ConA led to a dramatic drop in the percentage of ANAE⁺ cells after 48 to 72 hrs (80 % to 20 %) suggesting that this cytochemical marker is lost during blast transfonnation. Enrichment for OKT4⁺ and OKT8⁺ subsets, on the other hand, did not result in significantly altered percentage of ANAE + cells compared to the unseparated T cells as (OKT4⁺ were to 88 % and OKT8⁺ to 73 %) ANAE-positive.These data demonstrate that OKT antigens as well as Fc-IgG-R, Fc-IgM-R, and the ANAE spot are T-cell markers defining different aspects of T-cell function. This does not rule out that certain markers correlate to each other: their combined application may be useful further to dissect the T cell system.     

Reticular cells in peripheral lymphoid tissues express the phosphatidylinositol-linked BP-3 antigen.

The murine BP-3 antigen was initially described as a variably glycosylated cell surface protein of Mr 38,000 to 48,000 on lymphoid and myeloid cells. In the present experiments we found that this antigen is released from the surface of pre-B cells and macrophages by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), suggesting a glycosyl-phosphatidylinositol (GPI) linkage with the plasma membrane. When the tissue distribution of the BP-3-reactive cells was examined by immunohistology, high levels of the antigen were observed on brush borders of the intestinal epithelial cells, within collecting tubules of the kidney and on a subpopulation of reticular cells located on lymph nodes. Peyer's patches and the white pulp areas of the spleen. In contrast, reticular cells located in the thymus, bone marrow and splenic red pulp did not express the BP-3 antigen. Ontogenic studies revealed that BP-3 was expressed by the reticular cells in peripheral lymphoid tissues in the neonatal period near the time of lymphocyte immigration into these organs. BP-3+ reticular cells were observed in the collapsed periarterial lymphatic sheaths of adult mice depleted of T and B cells by cyclophosphamide treatment and in mice with severe combined immunodeficiency (scid), indicating that development of this reticular network is lymphocyte independent. The BP-3 antigen on the splenic reticular cells was also GPI anchored but its glycosylation pattern differed from that of the BP-3 molecules on pre-B cells. A specific subpopulation of reticular cells is thus marked by the BP-3 antigen, and the distribution and biochemical properties of the molecule make it an attractive candidate for a role in lymphocyte-stromal interactions in the peripheral lymphoid tissues.     

Identification of lymphocyte subsets in peripheral blood and thyroid gland in Graves' disease by alpha-naphthyl-acetate-esterase reaction

T lymphocyte subsets were prospectively examined in the peripheral blood and thyroid aspirates of 10 patients with hyperthyroid Graves' disease before and after treatment with methimazole and attainment of euthyroidism. T lymphocyte subsets were identified with monoclonal antibodies and pattern of alpha-naphthyl-acetate esterase (ANAE) staining pattern in the case of peripheral blood and ANAE staining pattern with thyroid aspirate smears. Before treatment, OKT8+ lymphocytes were significantly decreased (18.4% +/- 4.8) (S. D.) in the patients compared to control (28.8 +/- 6.7%, p less than 0.05), the OKT4/OKT8 ratio was increased (2.92 vs 2.11). Percent OKT8+ lymphocytes were not different from the controls when the ten patients had been rendered euthyroid. ANAE mononuclear cells with a diffuse pattern (presumed suppressor cells) were 4.2% +/- 1.8 before treatment and 8.3 +/- 2.4 (p less than 0.05) after treatment and 11.5% +/- 2.2 in controls. ANAE mononuclear cells with diffuse pattern represented 4.2% +/- 1.8 of the mononuclear cells infiltrating the thyroid gland of untreated patients and rose to 8.3% +/- 2.4 after the patients had become euthyroid. ANAE negative cells (B cells and some T cells) were increased in the thyroid of untreated patients. It is concluded that mononuclear cells with presumed suppressor T cell phenotype are decreased in the blood and thyroid glands of patients with active Graves' disease and that this defect is corrected when euthyroidism has been established.