Acid Α-naphthyl Acetate Esterase Research Articles

Suppressor cells of the human NK activity: Characterization of the cells and mechanism of action

The surface marker characteristics and mechanism of action of small- to medium-sized NK suppressor lymphocytes, which can be found in both umbilical cord blood and adult peripheral blood, have been studied. Evidence suggestive of T-cell origin of the lymphocytes consisted of E-rosette formation, reactivity with OKT3 monoclonal antibody, and dot-like acid α-naphthyl acetate esterase (ANAE) staining pattern typical of T cells. Furthermore, no reactivity was seen with OKT6 and OKM1 monoclonal antibodies and the presence of intracytoplasmic immunoglobulin was excluded by indirect immunofluorescence microscopy, making the involvement of monocytes, B cells, and thymocytes less likely. As regards the mechanism of action, the role of prostaglandins was unlikely since indomethacin had no effect on the level of suppression. The role of soluble mediators was further examined by blocking cell secretion with monensin. In these experiments monensin treatment of the suppressor cells did not unwind suppression, suggesting that mechanisms other than secretion of suppressive factors were operative. The importance of cell-to-cell contact was demonstrated by the following observations: (i) A short contact of effector lymphocytes with suppressor lymphocytes, followed by their physical separation, resulted in decreased cytotoxic activity of the effector cells, (ii) Suppression could be mediated through Nuclepore filters, which allowed cell processes to pass through the filter, but not through filters which did not allow cell-to-cell contact. The suppressor cells were resistant to irradiation (2500 rad) and treatment with dexamethasone and puromycin. Viable cells were not needed, since paraformaldehyde-fixed suppressor cells could also mediate inhibition of K562 killing.

Inflammatory cells in sarcoid granulomas detected by monoclonal antibodies and an esterase technique

Inflammatory cells in situ in Kveim reaction papules were identified in 15 patients with avidin-biotin-peroxidase complex and biotin-avidin-peroxidase methods for surface epitopes and with a simultaneously capturing azo dye method for cytoplasmic acid α-naphthyl acetate esterase (ANAE). The spatial relationship of cells in granulomas indicates a concentric arrangement. Endogenous peroxidase-negative, ANAE+, but OKIa-negative immunoincompetent epithelioid cells were in the center. T3+, ANAE+ T lymphocytes formed 60–80% of all cells in the lymphocyte mantle surrounding the epithelioid core. T4 T8 was 2:1. Equal proportions (5–15%) of Ia+ lymphocytes and M pattern ANAE+, endogenous peroxidase-positive mononuclear phagocytes on the one hand and T3+ and T pattern ANAE+ cells on the other in individual patients indicate that the proportion of activated T blasts in situ was less than 15%. The close contacts between different immunocompetent cells in the periphery indicates this as the site for cellular interactions.

Characterization by Acid α-Naphthyl Acetate Esterase Staining of the Spinal Cord Cellular Infiltrate in the Acute and Relapse Phases of Chronic Relapsing Experimental Allergic Encephalomyelitis

This chapter discusses chronic relapsing disease. A histopathological examination of the central nervous sytem (CNS) at different stages of the chronic relapsing model has shown variations in the inflammatory reaction. The chronic relapsing disease in guinea pigs displays a relapsing and remitting course together with the development of demyelinating plaques. The infiltrates are largely meningeal in the acute phase, becoming parenchymal and associated with demyelinating plaques during established relapses. Lymphocytes, macrophages and plasma cells have been described in these lesions by morphological criteria. This chapter describes the characterization of guinea pig lymphocytes and macrophages by histochemical means. Studies on human lymphocytes have shown staining for acid a-naphthyl acetate esterase (ANAE) to be specific for a large proportion of T-lymphocytes. Monocytes (macrophages) also stain, but their esterase activity is inhibited by the addition of 10 mM fluoride ion (F-) to the staining medium. B-cells have been shown in man not to stain for ANAE.

The T lymphocyte content of the efferent lymphatics of the human palatine tonsil.

Human palatine tonsils have been studied by enzyme histochemical means for T lymphocyte marking enzymes (acid α-naphthyl acetate esterase and acid phosphatase). These enzymes show discrete dot like activity in T lymphocytes. An almost pure population of T cells (90–100% of cells present) is seen in the efferent lymphatics of all of the 12 tonsils studied. These findings are of probable immunological significance.

Acid α-naphthyl acetate esterase (ANAE) in human B cells: Correlation of expression with stages of B-cell differentiation

Normal and malignant cells of the B cell lineage were examined for their expression of acid α-naphthyl acetate esterase (ANAE) and correlation of ANAE expression with cytoplasmic immunoglobulin (CIg), a marker which defines plasma cells. Resting peripheral blood B cells are ANAE −. In contrast, the majority, but not all, benign plasma cells and the malignant plasma cells of Waldenstrom's macroglobulinemia and multiple myeloma express ANAE in a distinctive staining pattern, i.e., multiple, variably sized nodules of reaction product in the paranuclear Golgi zone, which is distinguishable from that of monocytes and T cells. A variable proportion of pokeweed mitogen-stimulated B cells and the cells in each of three immunoglobulin-secreting B cell lines also expressed ANAE. In these instances the percentage ANAE + cells exceeded the percentage CIg + cells. A variable proportion of the neoplastic cells, 21–76%. in 4 of 25 cases of B-chronic lymphocyte leukemia (CLL) unassociated with monoclonal serum proteins and 2 of 4 cases associated with monoclonal serum protein production expressed ANAE in a staining pattern similar to that of plasma cells but with weaker intensity. B-CLL and plasma cell ANAE activity was inhibited by sodium fluoride, similar to monocyte and dissimilar from T cell ANAE activity. The malignant cells of acute myelogenous and lymphoblastic leukemia were ANAE −. These findings suggest that B cells express ANAE as they differentiate into plasma cells and/or are activated. Moreover, variable ANAE expression is another example of inter- and intratumor heterogeneity displayed by certain B cell malignancies.

Enzymecytochemical heterogeneity of human chronic T-lymphocytic leukemia as demonstrated by reactivity to dipeptidylaminopeptidase IV (DAP IV; EC 3.4.14.4)

The observation that the dipeptidylaminopeptidase IV (DAP IV; EC 3.4.14.4) occurs exclusively in Tμ lymphocytes, stimulated the following study on cases of chronic lymphocytic leukemias. Thirty cases of human chronic lymphocytic leukemia were subjected to the DAP IV reaction in addition to the usually applied cytochemical and immunological tests (acid α-naphthyl acetate esterase, acid phosphatase, rosetting with sheep erythrocytes and surface Ig). DAP IV activity was measured directly in normal granulocytes, monocytes, B and T lymphocytes as well as in lymphocyte suspensions of leukemic cases. On a cytochemical level granulocytes, monocytes, B lymphocytes and all 24 cases of B-CLL were found to be DAP IV negative, though some of the latter showed positive reactions for AcE and AcP. From the six cases of T-CLL, five were positive to DAP IV cytochemically. No discrepancies were observed between cytochemical and biochemical results. It is concluded that DAP IV is a reliable and easy to perform marker for Tμ lymphocytes and their neoplasias. The results have been interpreted as further evidence for a clonal nature of T-cell neoplasias.

Polymorphism and antigenic specificity of monocytic acid esterase (EC 3.1.1.6)

With the aim to provide a biochemical or immunological marker for human blood monocytes, their differentiational derivatives, and neoplastic variants the polymorphism of acid α-naphthyl acetate esterase was investigated. Using an isoelectric focusing technique it could be shown that the five enzyme variants of normal human blood monocytes, which do not occur in other human blood cell types, were regularly detectable in human peritoneal and alveolar macrophages. Antisera, raised against isoenzymes of monocytic acid esterase in rabbits, reacted in all cases with every five isoenzymes of blood monocytes; no cross-reactivity was observed with the isoenzymes of acid esterase obtained from purified human granulocytes, peripheral lymphocytes, B lymphocytes, and thymocytes as shown by immunoprecipitation. The results indicate that monocytic acid esterase and its variants could be regarded as a biochemical as well as immunological marker for this cell line.

T lymphocytes and mononuclear phagocytes in the skin infiltrate of systemic and discoid lupus erythematosus and Jessner's lymphocytic infiltrate.

T Lymphocytes and mononuclear phagocytes were measured quantitatively with the histochemical acid alpha-naphthyl acetate esterase (ANAE) method from paraffin sections of skin affected by systemic and discoid lupus erythematosus and by Jessner's lymphocytic infiltrate. The composition of the patchy cutaneous mononuclear cell infiltrates was similar in these three disorders.

Evaluation of acid α-naphthyl acetate esterase activity as a marker of T lymphocytes in benign and neoplastic cutaneous infiltrates

Activity of the enzyme acid alpha-naphthyl acetate esterase (ANAE) in T lymphocytes and histiocytes in cutaneous tissue sections of patients with histologically confirmed lichen planus, chronic discoid eczema and mycosis fungoides was compared with results obtained using an in situ immunohistochemical technique employing an antiserum against the human T lymphocyte surface antigen (HTLA). In lichen planus and chronic discoid eczema, most of the cells in the cutaneous infiltrate exhibited both positive ANAE activity and the presence of HTLA. In mycosis fungoides, cells identified as T lymphocytes by the presence of HTLA often showed no ANAE activity. ANAE activity appears to be a useful marker of T lymphocytes in benign cutaneous infiltrates but is less reliable in labelling abnormal T cells.

Spontaneous Cell-Mediated Cytotoxicity (SCMC) Associated with Lymphocytes Negative for Acid α-Naphthyl Acetate Esterase (ANAE) Activity

A modified histochemical procedure for nonspecific acid α-naphthyl acetate esterase (ANAE) activity in human lymphocytes was used to identify a subpopulation of E-rosette forming cells. Performing a one hour reaction at pH 6.5 the distinct dot-like staining pattern was almost exclusively observed on high affinity E-rosettes which sedimented readily in a regular Ficoll-Metrizoate gradient. By combining latex phagocytosis with staining for ANAE activity, a clear-cut distinction between mononuclear phagocytes and lymphocytes could be made. An attempt was undertaken to relate the ANAE marker on human lymphocytes to their functional capacity in spontaneous cell-mediated cytotoxicity (SCMC) reactions. Using as targets two allogeneic (K562,IGR3) and a xenogeneic cell line (L1210) it could be clearly demonstrated that high SCMC activity is mediated by ANAE negative mononuclear cells, whereas enrichment for ANAE positive lymphocytes resulted in a loss of SCMC.

Composition and in vitro cytotoxicity of cellular infiltrates in rejecting human kidney allografts

Four human renal allografts were removed during the primary acute rejection because of transplant rupture. The transplants were perfused from blood and disaggregated into single cell suspension with collagenase-DNAse. The low red cell/white cell ratio in the dispersate, compared to blood, demonstrated that blood contamination of the dispersates was minimal. Cytological and subclass analysis of the infiltrating ceils was performed from cytocentrifuged preparations of the initial dispersate. Approximately 26% of the allograft-infiltrating cells were tissue macrophages, 24% lymphocytes, 19% monocytes, and 18% polymorphonuclear leukocytes. Lymphoblasts (7%) and plasmablasts plus plasma cells at various stages of maturation (5%) were also present in the infiltrates. Seventy-four percent of the infiltrating lymphocytes were SRBC-binding and acid α-naphthyl acetate esterase (ANAE) marker-expressing (T) cells, and 14% SIg-carrying (B) cells. Most (83%) of the infiltrating macrophages, 47% of the infiltrating monocytes and 23% of the infiltrating lymphocytes expressed the Fc-receptor for IgG. The infiltrating cells and transplant parenchymal cells were fractionated apart by 1 g velocity sedimentation. The infiltrating cells and blood mononuclear leukocytes of the transplant recipients were then tested for in vitro cytotoxicity to cultured donor spleen-derived and irrelevant macrophages, and to the parenchymal cells of the transplant. The infiltrating cells were far more cytotoxic to donor-derived macrophages than were blood leukocytes, and the cytotoxic effect was specific. In contrast, the blood leukocytes and the infiltrating cells killed extremely weakly, if at all, the (relevant) transplant parenchymal cells. This suggests that other mechanisms than direct contact-mediated cellular cytotoxicity are partially responsible for the deterioration of the graft function during allograft rejection.

Distribution of four subclass-specific lymphocyte markers in man.

A report is given of the relative and absolute distribution of lymphocytes carrying three T or B cell specific markers in cord blood; in children and in the blood of healthy adult human subjects. The determinations were performed from stained cytological cell smears under conditions where no subclass specific lymphocyte selection or depletion takes place. In adult blood 78 896 (S. D.) (1.2 0.4 109/1) of lymphocytes expressed the acid α‐naphthyl acetate esterase (ANAE) marker. The percentage distribution of the ANAE marker‐carrying lymphocytes was stable in different age groups. The absolute values in cord blood and in young children were approximately three times higher than in adults. A somewhat smaller fraction of adult lymphocytes (5314%; 1.0 0.4 109/1) formed rosettes with 2‐amino‐ethylisothiouronium bromide‐hydrobromide (AET) sensitized sheep erythrocytes (AET‐SRBC). Although the relative distribution of AET‐SRBC rosette‐forming lymphocytes was slightly lower in children than in adults, the absolute numbers were approximately two to three times higher. The number of surface Ig‐carrying lymphocytes, as detected by rosette formation with Staphylococcus aureus strain Cowan I (StaCw) bacteria in adolescent and adult blood was 19 11 % (0.4 0.2 109/1). The relative number of surface Ig‐carrying lymphocytes varied very little in the different age groups. The absolute numbers in cord blood and in small children were approximately twice as high as in adults. The distribution of lymphocytes carrying the fourth marker, surface receptor for IgG, was investigated by antibody‐coated human erythrocytes (EA). In adults, the number of lymphocytes forming rosettes with EA was 30 10 % (0.7 0.3 109/1). Although the relative values were somewhat lower in children, the absolute values were approximately twice as high as in adults.

The demonstration of acid α-naphthyl acetate esterase activity in human lymphocytes: Usefulness as a T-cell marker

The histochemical demonstration of nonspecific acid α-naphthyl acetate esterase (ANAE) activity was evaluated as a T lymphocyte marker primarily with the sheep erythrocyte (E) assay. A distinctive staining pattern characterized T lymphocytes which could be readily distinguished from monocyte staining. The percentage of E + and ANAE + lymphocytes was nearly always comparable in the peripheral blood and lymphoid tissue from normal and selected patients, including those with acute and chronic lymphocytic leukemia. Divergences were noted in certain other tissues including spleen and thymus. Certain mitogen-stimulated cells lost their ANAE activity while retaining their ability to form e n rosettes. Atypical and variable staining patterns were observed in established lymphoid cell lines. The histochemical demonstration of ANAE is simple and reproducible; preparations may be counterstained for cytomorphologic detail and mounted as a permanent record. Certain disadvantages are discussed. The method represents a practical alternative to E rosette assays. It is particularly well suited for certain routine laboratories.

Immunohistological studies on intracerebral transplantation of transplantable malignant glioma of C57BL/6J mice (author's transl)

Transplantable gliomas of C57BL/6J mice were used in order to investigate the influence of immunological resistance against tumors transplanted in the right parietal lobe. Immunization was induced by subcutaneous inoculation of approximately 1 mm3 of tumor grafts, irradiated by 3, 000 watt.sec/cm2 ultrasound or 3, 000 rad X-ray, once a week for 2 weeks to normal animals. Immunization was assured by tumor rejection or no growth 2 to 3 weeks after challenge of non-treated tumor implants. The rates of immunization against tumor were 67% by ultrasound irradiation and 45% by X-ray irradiation. Passive transfer of resistance against the tumor was evaluated by IV injection of 1 × 108 spleen cells from immunized animals to normal isologous animals. The result showed a 40% rate of transfer. The rate of tumor growth transplanted in the brain was 71 % in immunized mice induced by subcutaneous inoculation with no significant difference from normal mice (81%). However, survival intervals after transplantation were significantly longer (P<0.01), ranging from 17.6 to 23.7 days. The tumor was more localized and showed more degeneration. Lymphocytic infiltration into the brain tumor was much less pronounced than subcutaneous tumor in immunized animals and there was no marked glial reaction. Focal lymphocytic accumulations were sometimes observed in perivascular spaces and in the meninges, but there were no clear differences from non-immunized tumor-bearing mice. The population of T-cells in the spleen and the mesenteric lymphnodes was evaluated by acid α-naphthyl acetate esterase (ANAE) stain in imprint preparations and in freshly snap-frozen (-80°C) and cryostat-cut specimens of the spleen, lymphnodes and brain tumors. ANAE stain reported by Mueller in 1975 demonstrated one or two positive dots in T-cells. T-cells in the spleen and lymphnodes increased but only a few were detected in the brain tumor of the immunized animals. Direct immunofluorescent studies with FITC-labelled anti-mouse Ig antibodies were applied to freshly snap-frozen and cryostatcut specimens. As a result, Ig-cotaining cells infiltrated the tissues around the subcutaneous tumor while a few penetrated the tumor tissues. Fluorescence was not marked on the cytoplasmic membrane of brain tumor of immunized animals but was positive in those of nonimmunized animals and was more marked in those animals which showed tumor growth in spite of induction of immunization, suggesting that the blocking factor described by Hellstrom was involved.

T Cells as Marked with Acid α-Naphthyl Acetate Esterase Staining in Secretory Otitis Media

Non-specific acid alpha-naphthyl acetate esterase (ANAE) activity of the lymphocytes was investigated in smears made of middle ear secretions and peripheral blood from 61 children with secretory otitis media. On the average, 20.1% (right ear) and 2.1% (left ear) of the lymphocytes in the ear samples showed distinctive ANAE-positive spots in their cytoplasms, by the method used. The mean percentage of ANAE-positive cells in the blood was 47.8 and it was significantly (P less than 0.001) higher than the respective percentages of the ear samples. In normal childrne, 54.9% of the lymphocytes, on the average, were ANAE-positive. This difference in blood between patients and control group was significant (P less than 0.001). Consequently, the present study suggests that the relationship of T- and non-T-lymphocytes, as a marked with ANAE, are disturbed in children suffering from secretory otitis media. The importance of this finding and the validity of the method are discussed.