Clin. exp. Immunol. (1977) 30, 289-298.
Identification of human mononuclear leucocyte populations by esterase staining D. A. HORWITZ, A. C. ALLISON, P. WARD & NANCY KIGHT Division of Cell Pathology, Clinical Research Centre, Watford Road, Harrow, Middlesex, and The Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville, Virginia, U.S.A. (Received 26 May 1977)
SUMMARY
Histochemical staining for cx-naphthyl (non-specific) esterase has been employed to define subpopulations of human peripheral blood lymphocytes and monocytes. To define optimal conditions for staining, various fixatives, incubation times and cell preparations were compared. The great majority of human blood lymphocytes were found to have discrete granules of reaction product. More than 80% of T lymphocytes separated by E rosetting are esterase-positive whereas non-T lymphocytes are esterase-negative. Lymphocytes transformed by polyclonal mitogens lose their esterase-staining granules, which suggests that immature T cells are esterase-negative. Most blood monocytes show a diffuse cytoplasmic esterase reaction product and are phagocytic. However, about 20% of diffusely stained cells are not phagocytic. When leucocytes are cultured for 24 to 48 hr, the total number of esterase-positive cells increases and the great majority are phagocytic. This is interpreted as maturation of precursors into mature esterase-positive phagocytic monocytes. When cultured for longer periods, some lose phagocytic capacity and acquire the characteristics of secretory cells. Esterase-staining of lymph node sections allowed the identification of T- and B-dependent areas as well as macrophages related to sinuses. The esterase-staining technique could play a useful role in clinical and experimental immunology. INTRODUCTION During the past few years there has been increasing interest in defining populations of mononuclear leucocytes in human and animal peripheral blood and lymphoid organs. The original distinction was made between lymphocytes and cells ofthe mononuclear phagocyte lineage (monocytes and macrophages). Later, T and B lymphocytes were defined (Greaves, Owen & Raff, 1973) and it has become apparent that subpopulations of each of these exist. Evidence of a third population, morphologically indistinguishable from lymphocytes, has accumulated (Fr0land & Natvig, 1973; Horwitz & Lobo, 1975). These have been named L lymphocytes because of labile IgG determinants bound on their surface by highly avid Fc receptors. Currently used methods for characterizing these leucocyte populations, such as rosetting and membrane immunofluorescence, are laborious and time-consuming. There is a real need for a simple procedure to define leucocyte populations for clinical purposes. Histochemical demonstration of nonspecific esterase promises to meet this need. For some time histochemical reactions for esterase, using as the substrate oc-naphthyl acetate, has been employed for the identification of mononuclear phagocytes (Schmalzl & Braunsteiner, 1968; Yam, Li & Crosby, 1971). More recently Mueller et al. (1975) have reported localized granules of esterase reaction product in mouse lymph node T lymphocytes, whereas cells of the B-lymphocyte lineage were Correspondence: Dr D. A. Horwitz, Division of Cell Pathology, Clinical Research Centre, Watford Road, Harrow, Middlesex.
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esterase-negative. Ranki, Totterman & Hiyry (1976) used the esterase method to distinguish T lymphocytes identified by E-rosetting from B lymphocytes identified by rosetting with Staphlococcus aureus strain Cowan 1. These workers did not mention additional populations, such as L cells, nor did they comment on the usefulness of the esterase-staining method for identifying cells of the monocyte lineage, some of which are not phagocytic. No studies have been reported on human lymphoid organs. Because of the potential usefulness of the esterase method, we have undertaken systematic studies of human peripheral blood smears, purified mononuclear cell populations and sections of human lymph nodes. Techniques have been modified to ascertain the optimal conditions for differentiating between different populations of leucocytes. Moreover, the staining patterns of unstimulated lymphocytes in short-term cultures and cells exposed to T- and B-lymphocyte mitogens has been investigated. MATERIALS AND METHODS Reagents. Solutions for fixation and preparation of tissue sections. (a) Buffered formalin-acetone, pH 6-6: 20 mg Na2HPO4 was mixed with 100 mg KH2PO4, 30 ml H20, 45 ml acetone and 25 ml of 40% formaldehyde (Yam, Li & Crosby, 1971). (b) 10% neutral formol-calcium: 10 ml 40% formaldehyde was added to 1 1 mg anhydrous calcium chloride, 90 ml distilled water and adjusted to pH 7 0 with 1 N NaOH. (c) Buffered glutaraldehyde: 0-1 M phosphate buffer, pH 7-4, was added to glutaraldehyde to give a 2.5% solution. (d) Holt's gum sucrose 0-88 M: 30 g sucrose and 1 g gum acacia were mixed in the dry state and dissolved in distilled water to give a final volume of 100 ml (Pearse, 1968). (e) Gelatine-formaldehyde: 50 ml 1% gelatine was added to 50 ml 2% formaldehyde. Esterase staining. (a) Pararosanilin stock solution: 2 g pararosanilin hydrochloride (Hopkin & Williams, Chadwell Heath, Essex, U.K.) and 50 ml 2 N hydrochloric acid were heated gently, cooled to room temperature and filtered (Davis & Ornstein, 1959). (b) Incubation mixture, pH 6-1: the esterase substrate should be prepared immediately before use by dissolving 4 mg a-naphthyl acetate (Sigma Chemical Company, London) in 0 5 ml ethylene glycol monomethyl ether (Koch-Light Laboratories, Colnbrook, Buckinghamshire, U.K.) and mixed with 8-9 ml M/15 phosphate buffer, pH 7-6, and 0-6 ml hexazotized pararosanilin. The solution should not require filtration. (c) Hexazotized pararosanilin: 0 3 ml pararosanilin stock solution was mixed with 0-3 ml of freshly made 4% sodium nitrite and allowed to stand for 1 min. (d) Methyl green counterstain: 2 g methyl green was dissolved in 100 ml of heated distilled water. Chloroform-soluble material was extracted and the solution heated to 60'C to evaporate residual chloroform (Bancroft, 1975). (e) Polyclonal mitogens: phytohaemagglutinin M (PHA; Difco Laboratories, Detroit, Michigan), concanavalin A (Con A; Pharmacia, Uppsala, Sweden), and pokeweed mitogen (PWM; Grand Island Biological Co., Grand Island, N.Y.).
Proceedures. Blood and mononuclear cell smear staining. Blood was collected from each donor by venepuncture and a drop smeared onto a glass slide for esterase-staining. The remainder was heparinized (50 u/ml), diluted 1: 3, layered on FicollTriosil and centrifuged at 400 g at room temperature for 35 min. The mononuclear cells were harvested, washed three times, adjusted to 106/ml and 3 to 6 drops were pelleted onto 22 x 22 mm cover slips in a Shandon-Elliot cytocentrifuge (Shandon Scientific Company, London). The cover slips were air-dried and immediately fixed in 4VC buffered formalin-acetone for 30 sec, washed in three baths of distilled water and air-dried for at least 30 min. Both the blood smears and cytocentrifuge cover slips were stained in freshly prepared unfiltered incubation medium at room temperature for 45 min unless stated otherwise. They were rinsed free of visible deposits which sometimes formed during staining. The smears were counterstained in 2% methyl green for 5 min, rinsed in three changes of distilled water and air-dried. They were then rinsed in xylene and mounted with Ralmount (Raymond A. Lamb, London). Cell cultures. 5x 106 mononuclear cells were suspended in 5 ml RPMI 1640 containing 15% bovine serum, 2 mm glutamine and 50 pg/ml gentamycin (Flow laboratories, Irvine, Scotland). They were cultured in 30 ml tissue-culture flasks (NUNC A/S Roskilde, Denmark) for 4, 24, 48 and 72 hr. 0 5 ml (1:10) phytohaemagglutinin, 5 pg/ml concanavalin A and 0 5 ml pokeweed mitogen were added to some cultures. Five drops of polystyrene particles (Bacto Latex 0-81 pm, Difco, Detroit, Michigan) diluted 1:10 were added for the last 4 hr of culture. The cells were washed twice, an aliquot counted with a Coulter counter, viability estimated by trypan blue exclusion and cytocentrifuge smears were prepared. Tissue sections. Human lymph node specimens were fixed overnight in 10% 4VC neutral formol calcium and transferred to Holt's gum sucrose for at least 24 hr at 4VC. They were then washed in running water for 10 min, frozen in liquid nitrogen and 9 pm sections cut with a cryostat. The sections were mounted on cover slips pretreated with gelatine-formaldehyde (Pearse, 1968), allowed to dry for 1 hr at room temperature and incubated with the esterase reaction mixture for 40 min. After washing and counterstaining with methyl green, the sections were washed, dehydrated with graded alcohols, rinsed in xylene and mounted in Ralmount.
RESULTS Technical considerations Blood smears and cytocentrifuge smears were fixed and stained at various times after they were prepared. Blood smears could be stored for 2 to 3 weeks without loss of staining properties. Enzyme
gSj~ ~ '.|i ',:_. Mononuclear leucocyte populations
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activity appeared to decrease, however, in cytocentrifuge smears of mononuclear cells. Best results were obtained when the mononuclear cells were fixed and stained shortly after the smears were prepared. Several procedures for cell fixation were compared to establish which was most satisfactory. Fixation with cold formalin-acetone for 30 sec resulted in good preservation of cell morphology and optimal esterase staining. Fixation in cold formal-calcium for 10 min (Mueller et al., 1975) also resulted in good esterase staining, but with considerable loss of morphological detail. Fixation in buffered glutaraldehyde at 4VC for 10 min (Mueller et al., 1976) resulted in loss of both esterase reactivity and morphological detail. Cold formalin-acetone is, therefore, the procedure of choice. Cytocentrifuge smears were stained for various times. Large mononuclear cells with the morphological features of monocytes were faintly positive at 20 min, and by 45 min were diffusely stained with reddishbrown reaction product. After 20 min 30 to 40% of lymphocytes developed one to four discrete granules of reaction product. After 45 min, 50 to 70% of lymphocytes had developed these punctate granules, usually close to the plasma membrane. At 90 min, the proportion of lymphocytes with discrete granules remained constant, but other lymphocytes developed ill-defined areas of reaction product (smudges) within the cytoplasm. After 3 to 5 hr of incubation with substrate, it became difficult to distinguish positive from negative lymphocytes, and all cells acquired a 'scorched' appearance. Since positive and negative cells could be distinguished optimally after 45 min of incubation, this time was selected for further studies. Examples of positive monocytes, together with positive and negative lymphocytes, in cytocentrifuge and blood smears are shown in Fig. 1. !~~~~l
- r Ii B ::i_*l fs
FIG. 1. Esterase-staining patterns in blood smears and cytocentrifuge mononuclear preparations. (a) Illustrates a diffusely staining monocyte, a lymphocyte with a discrete granule and negative lymphocytes; (b) shows a lymphocyte with one granule and a negative polymorphonuclear leucocyte; and (c) (cytocentrifuge smear) shows a diffusely positive monocyte which has phagocytosed latex particles, also several lymphocytes with one or more sharply defined granules of reaction product and several negative lymphocytes. (Magnification x 540.)
Staining patterns oflymphocytes Counts of granular and diffuse esterase-positive and of esterase-negative cells made in cytocentrifuge preparations of peripheral blood mononuclear cells from healthy subjects are shown in Table 1. The diffusely stained cells, which were of the mononuclear phagocyte lineage (see below), were subtracted from the total cell counts in order to determine the proportion's of lymphocytes with granular esterasestaining (Table 2). These results were compared with the percentage of E rosette-forming T cells in the same mononuclear suspension and the percentage of granular-staining lymphocytes in blood smears. Table 2 reveals that the great majority of blood lymphocytes were positive for both E-rosetting and granular-staining. The percentages of granular-staining cells in blood smears and cytocentrifuge preparations were similar. Although in some subjects the percentage of granular-staining cells approached that of E rosette-forming cells, in others the number of esterase-positive cells was 10 to 20% lower. Evidently most, but not all, T lymphocytes give a localized esterase reaction product. The correspondence between these two populations was confirmed by separating E-rosetting cells from the whole population. 80 to 90% of T lymphocytes, purified as described above and cytocentrifuged onto cover slips, showed the characteristic esterase-staining (Fig. 2b). Less than 8% of lymphocytes in B and L preparations showed granular staining, and this was due to residual T lymphocyte contamination (Fig. 2a). These preparations contained 6 to 8% E-rosetting lymphocytes.
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TABLE 1. Esterase-staining of mononuclear cells from normal
subjects Positive (%) Granular pattern
Subject
61 62 67 52 66 52 61 62 69 66 61-8
J.D. D.H. J.W. J.C. H.E. A.K. L.B. (Initial) (Repeat)* A.T. (Initial) (Repeat)* Mean *
Diffuse pattern 19 11 13 35 16 28 19 10 20 21 19-2
Negative (0) 20 27 20 14 19 20 20 28 11 13
19'2
Repeat examination 3 weeks after the initial study.
TABLE 2. Comparison of T lymphocytes detected by the E-rosette method and esterase technique
Granular-staining lymphocytes (0) Subject J.D. J.W. A.T. L.B. A.K. A.N. T.A. H.E. J.E. Mean
E rosettes (0)
86 84 82 81 78 77 75 73 67 78-1
Blood smear 69 76 81 70 72 65 63 77 65 70 9
Mononuclear cells* 72 77 82 68 72 64 70 75 59 71-0
* Cytocentrifuge preparations of mononuclear cells obtained by Ficoll-Triosil density gradient centrifugation.
Mononuclear lymphocytes prepared by Ficoll-Triosil centrifugation were cultured for 72 hr and developed changes in esterase-staining patterns. The proportion of granular-staining lymphocytes declined during the first 2 days in culture, but thereafter increased (Fig. 3). Other lymphocytes were stimulated with PHA, Con A and PWM, and esterase-staining determined after 24, 48 and 72 hr of culture. These polyclonal mitogens did not alter the esterase activity in small lymphocytes, but the activity declined as lymphocytes enlarged and became immature blasts. Most transformed cells showed little or no staining (Fig. 4). In some cells clusters of very fine granules were found.
Staining patterns of mononuclear phagocytes A major advantage of the esterase technique is that it identifies cells of the mononuclear phagocyte lineage at a relatively early stage of differentiation. 19% of blood mononuclear cells showed diffuse esterase-staining. Approximately 20% of these cells in all normal subjects tested did not phagocytose
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FIG. 2. Examples of mononuclear populations stained for esterase. (a) Negative non-T cells separated by Ficoll-Triosil centrifugation of E rosette-forming cells; (b) positive T cells after red cells have been lysed by ammonium chloride. (c to f) Mononuclear phagocytes after short-term culture; (c) 4 hr, examples of diffusely stained cells, one of which has ingested latex particles while the other has not; (d) 24 hr, almost all diffusely positive cells are phagocytic; (e) 48 hr, shows the development of a subpopulation with a round, eccentrically placed nucleus and cytoplasm with increased intensity of staining; phagocytic capacity is reduced; and (f) 72 hr, two non-phagocytic cells, one of which has developed numerous secretory granules; these are easily distinguishable from the smaller, more sharply defined latex particles. (Magnification x 540.) 80
r
60 0
0'< c
.0 71i
40
20
0
I
4
24
48
72
Time (hr)
FIG. 3. Granular esterase-staining of mononuclear cells prepared by Ficoll-Tricsil density gradient centrifugation. Error bars denote s.e.m.
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FIG. 4. Lack of esterase-staining of lymphocytes transformed by concanavalin A after 72 hr of culture. Identical fields by bright field (a) and phase-contrast (b) microscopy. Two large immature blast-like cells are shown which are esterase-negative; a positive small lymphocyte and a phagocytic monocyte are included for comparison. (Magnification x 540.)
latex particles in dilute suspensions. Examples of these cells are shown in Fig. 2c. When the cells were cultured for 24 hr. more than 95% of the diffuse esterase-positive cells were phagocytic in each of five subjects (Fig. 2d). Another notable feature was that the total number of esterase-positive cells increased during the initial 48 hr of culture. This remains true even when corrections are made for non-viability (Fig. 5). The increase cannot be explained by cell multiplication because less than 02%/o of unstimulated 30 r-
01
20
M
0'
C3
(-)
10
0
4
48
24
72
Time (hr)
FIG. 5. Percentage and phagocytic capacity of diffuse esterase-stained mononuclear cells after short-term culture. (0) Indicate the proportion of diffusely positive cells and (o) indicate diffusely positive cells that have phagocytosed latex particles. The mean numbers of trypan blue viable cells from 4 to 74 hr were > 99Y%, 95%4, 85%4 and 78Y% respectively. Data points have been adjusted downwards to account for dead cells and therefore indicate an increase in the absolute number of diffusely positive cells.
ti. ~ ~W.;
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Mononuclear leucocyte populations
mononuclear cells in culture divide (Horwitz, 1972). Therefore, this observation implies maturation of precursor cells morphologically indistinguishable from lymphocytes into esterase-positive monocytes. At 48 hr and subsequently, the proportion of phagocytic diffusely stained cells declined and changes in monocyte morphology were evident. At the start of the culture period the cells were round and the nuclei had a typical kidney-shaped, indented or lobed appearance (Fig. 2c). After 48 hr of culture the intensity of esterase-staining had increased and many cells acquired an oval shape. The nuclei had likewise become oval and eccentrically placed in the cell (Fig. 2e and f). Some of the cells developed numerous refractile secretory granules (Fig. 2f). Possibly these changes represent a stage of differentiation towards a non-phagocytic cell, such as an epithelioid cell. Staining patterns of lymph nodes Sections of fixed lymph nodes showed well-defined areas of esterase reaction product. Intense diffuse staining was observed in macrophages lining the marginal sinus and cortical sinuses connecting with the more concentrated network of sinuses in the medulla (Fig. 6). T lymphocytes with granular esterasestaining were easily identifiable under the high power (magnification x 40). At low power, areas of esterase reaction product less intense than that lining the sinuses appeared as a distinct reddish blush in the section. These corresponded to the paracortical (T-dependent) areas of the nodes.
_
~~~~4
*|
:-'b4i
X
~~~~~~~~~~~~~~~i
FIG. 6. Esterase-staining of lymph node. (a) Lymphoid modules (low-power magnification), heavily stained macrophages line marginal sinuses and paracortical areas contain 'blush' of stain. (Magnification x 325.) (b) High-power magnification, many paracortical T cells with discrete granules surround area of esterase-negative B cells. (Magnification x 540.)
Clinical examples Two examples of the clinical usefulness of esterase-staining are shown in Table 3. The first is the rapid detection of increased numbers of cells of the mononuclear phagocyte lineage in patient G.A. More than one-half of his mononuclear cells showed diffusely positive staining. Some of these were small round cells which morphologically resembled lymphocytes. After overnight culture, however, 97% of the diffusely positive cells had phagocytosed latex particles. Secondly, in both cases of immunodeficiency syndromes there was marked discrepancy in the number of E rosette-positive cells and lymphocytes with granular esterase-staining. In patient C.C. the number of esterase-positive lymphocytes was less than 20% of the number of E-rosetting cells. These findings suggest that immature T cells lack esterase activity.
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TABLE 3. Examples of abnormalities detected by esterase staining T lymphocytes (%)t
Granular Monocytes (%)* E rosette-forming esterase-staining
Comments
Patient
Diagnosis
G.A.
Congenital hypogammaglobulinaemia
62
69
50
Marked increase in the percentage of blood monocytes. Only 72% of T cells were esterase-positive. More than 80% of T cells in normals were esterase-
C.C.
Congenital hypogammaglobulinaemia
29
67
11
Marked decrease in esterase-positive T cells. Only 16% were positive
positive
* These are diffuse esterase-staining, expressed as percentage monocyte population.
t Expressed as percentage of total lymphocytes.
DISCUSSION Previous investigators had shown that esterase-staining could be used to identify either monocytes (Schmalzl & Braunsteiner, 1968) or a subclass of lymphocytes with a granular reaction product (putative T lymphocytes) (Mueller et al., 1975). The main purpose of this investigation was to develop a single method for the simultaneous demonstration of esterase-positive and -negative lymphocytes, and phagocytic or precursor cells of the mononuclear phagocyte lineage. For that purpose different methods of preparing, fixing and staining the cells have been compared. Both preparations from peripheral blood smears and cytocentrifuged Ficoll-Triosil mononuclear cells gave good staining. Three major categories of cells were readily identified, and counts made on the two preparations from the same individual showed excellent correspondence. There was no measurable loss of any cell population identifiable by the esterase technique during the preparation of mononuclear cells by density gradient centrifugation. Brown & Greaves (1974) reported that density gradient centrifugation decreased the percentage of E rosette-forming cells. Each preparation has advantages. The blood smear uses a small drop of blood which is dried in the conventional way and requires no special processing. Even the reagents for staining are commercially available (Tucker, Pierre & Jordan, 1977). Hence blood films can be used in looking for gross deficiencies. However, performing differential counts of enough cells to obtain statistically meaningful results is laborious and time-consuming. Density gradient preparations are widely used in clinical immunology laboratories, and it seems likely that this will become the routine method for preparing cells for esterase-staining, because of the high quality of the cells and the speed with which differential counts can be made. The great majority of blood lymphocytes were both E-rosetting and esterase-positive. The proportion of esterase-positive lymphocytes was usually less than that of E rosette-forming cells. Several explanations can be suggested. One possibility is a threshold effect on staining, that a proportion of T lymphocytes is not stained with the standard conditions used. However, this is unlikely because longer incubation results in weak reactions which may occur in all lymphocytes. A second possibility is that esterasenegative lymphocytes are the same as the subpopulation of T lymphocytes with Fc receptors. The Fc receptor-bearing subpopulation can comprise up to 20% of T cells) Ferrarini et al., 1975). This possiblity has not been excluded, but is unlikely. A third possibility is that esterase-negative E-rosetting cells are immature T lymphocytes. Consistent with this interpretation is the observation that mouse cortical thymocytes are nearly all esterase-negative (Mueller et al., 1975). Moreover, when T lymphocytes
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were transformed into immature blasts by polyclonal mitogens in culture, loss of esterase activity was observed. For these reasons, the latter explanation seems most likely and could have clinical application in revealing defects in T-lymphocyte maturation. This is illustrated by the two patients with immunodeficiency syndromes described in Table 3. The esterase technique also provides useful information about cells of the mononuclear phagocvte lineage. In addition to the well-known fact that monocytes are esterase-positive, it is possible to obtain information about their state of maturity. Approximately 20% of diffusely esterase-stained cells in peripheral blood from normal subjects were not phagocytic. When the cells were cultured for 24 to 48 hr the absolute number of esterase-stained cells increased, and 98% of these were phagocytic. These results suggest maturation of esterase-negative cells into cells with diffuse esterase-staining and the maturation of phagocytic capacity. After 72 hr in culture the proportion of phagocytic cells declined and of oval cells with the distinctive appearance of epithelioid cells increased. The cytoplasm of some of these cells was filled with secretory granules. Thus, different subpopulations of mononuclear phagocytes can be identified with esterase-staining. The present observations suggest that the proportion of monocyte-like cells in human blood is higher than generally supposed. This proportion may be further increased in patients with some infections, inflammatory diseases and malignant states. For example, in patients with systemic lupus erythematosus 40 to 50% of mononuclear cells are blood monocytes, as shown by acridine orange vital fluorescence, and this finding explains, at least in part, the decreased mitogenic reactivity (Horwitz & Garrett, 1977). Moreover, patients with systemic lupus erythematosus, other connective tissue disorders and infections also have increased numbers of monocyte precursors incorporating thymidine into DNA (Horwitz, 1972). In general, the esterase technique can provide useful information, not only about the total number of monocytes and T lymphocytes in the circulation, but also their degree of maturity. Esterase-negative cells include granulocytes (Yam et al., 1971), B lymphocytes and lymphocytes with high-affinity Fc receptors for IgG, termed L lymphocytes (Horwitz & Lobo, 1975). Even these may show weak reaction products after prolonged incubation with substrate. Besides being applicable to cell suspensions, the esterase technique can be used on sections of lymph nodes and other tissues. It provides a simple and reliable technique for demonstrating diffusely stained macrophages and granular-staining mature T lymphocytes. These results appear be to applicable to humans and several species of experimental and domestic animals. The identification of these cells without the need for specific antisera and without the uncertainties of in situ resetting is a considerable technical advance. It seems likely that the esterase technique will be applied routinely in many clinical and experimental laboratories. We are indebted to Dr D. Webster and Dr G. Slavin for providing blood samples and lymph node specimens. This work was supported by the John A. Hartford Foundation grant 74466, and United States Public Health Service grant 11766. Dr Horwitz is a recipient of the Clinical Scholar award from the Arthritis Foundation. REFERENCES
BANCROFT, J.D. (1975) Histochemical Techniques, 2nd edn, p. 260 and p. 322. Butterworths, London. BROWN, G. & GuAvEs, M.F. (1974) Enumeration of absolute numbers of T and B lymphocytes in human blood. Scand. 5. Immunol. 3, 161. DAVIS, B.J. & ORNSTEIN, L. (1959) High resolution enzyme localization with a new diazo reagent 'hexazonium pararosaniline'. 5. Histochem. Cytochem. 7, 297. FERRARINI, M., MORETTA, L., ABRILE, R. & DURANTE, M.L. (1975) Receptors for IgG molecules on human lymphocytes forming spontaneous rosettes with sheep red cells. Europ. 5. Immunol. 5, 70. FRoLAND, G.F. & NATVIG, J.B. (1973) Identification of three different lymphocyte populations by surface markers. Transplant. Rev. 16, 114.
GREAvEs, M.F., OWEN, J.J.T. & RAFF, M. (1973) T and B Lymphocyte Origins, Properties and Role in Immune Responses. Excerpta Medica, Amsterdam. HORWITZ, D.A. (1972) The development of macrophages from large lymphocyte-like cells in the blood of patients with inflammatory diseases. J. clin. Invest. 51, 760. HORWITZ, D.A. & GARRETT, M.A. (1977) Mitogenic reactivity of human lymphocytes. I. Decreased responsiveness in systemic lupus erythematosus, rheumatoid arthritis and scleroderma: technical considerations. Clin. exp. Immunol. 27, 100. HORWITZ, D.A., & LOBO, P.I. (1975) Characterization of two populations of human lymphocyte-bearing immunoglobulins. J. clin. Invest. 56, 1464. MUELLER, J., BRUN DEL RE, G., BUERKI, H., KELLER, H.U.,
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HESS, M.W. & COTTIER, H. (1975) Nonspecific acid esterase activity: a criterion for differentiation of T and B lymphocytes in mouse lymph nodes. Europ. 5. Immunol. 5, 270. MUELLER, J., KELLER, H.U., BRUN DEL RE, G., BUERKI, H. & HESS, M1.W. (1976) Nonspecific esterase activity in T cells. Immune Reactivity of Lymphocytes (ed. M. Feldman and A. Globerson), p. 117. Plenum Publishing Corporation, New York. PEARSE, A.G.E. (1968) Histochemisiry, Theoretical and Applied, 3rd edn, Vol. 1., p. 602 and p. 728. Churchill, London. RANKI, A., TOTTERMAN, T.H. & HAYRY, P. (1976) Identi-
fication of resting human T and B lymphocytes by acid anaphthyl acetate esterase staining combined with rosette formation with Staphylococcus aureus strain Cowan I. Scand. 7. Immunol. 5, 1129. SCHMALZL, F. & BRAUNSTEINER, H. (1968) Cytochemische Darstellung von Esterasere-aktivitat in Blut - und Kncckenmarkszellen. Kim'. Wschr. 46, 642. TUCKER, S.B., PIERRE, R.V. & JORDAN, R.E. (1977) Rapid identification of monocytes in a mixed mononuclear cell preparation. 3. immunzol. Methods, 14, 267. YAM, L.T., Li, CY. & CROSBY, W.H. (1971) Cytochemical identification of monocytes and granulocytes. Am. 7. Clin. Path. 55, 283.
Identification of human mononuclear leucocyte populations by esterase staining D. A. HORWITZ, A. C. ALLISON, P. WARD & NANCY KIGHT Division of Cell Pathology, Clinical Research Centre, Watford Road, Harrow, Middlesex, and The Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville, Virginia, U.S.A. (Received 26 May 1977)
SUMMARY
Histochemical staining for cx-naphthyl (non-specific) esterase has been employed to define subpopulations of human peripheral blood lymphocytes and monocytes. To define optimal conditions for staining, various fixatives, incubation times and cell preparations were compared. The great majority of human blood lymphocytes were found to have discrete granules of reaction product. More than 80% of T lymphocytes separated by E rosetting are esterase-positive whereas non-T lymphocytes are esterase-negative. Lymphocytes transformed by polyclonal mitogens lose their esterase-staining granules, which suggests that immature T cells are esterase-negative. Most blood monocytes show a diffuse cytoplasmic esterase reaction product and are phagocytic. However, about 20% of diffusely stained cells are not phagocytic. When leucocytes are cultured for 24 to 48 hr, the total number of esterase-positive cells increases and the great majority are phagocytic. This is interpreted as maturation of precursors into mature esterase-positive phagocytic monocytes. When cultured for longer periods, some lose phagocytic capacity and acquire the characteristics of secretory cells. Esterase-staining of lymph node sections allowed the identification of T- and B-dependent areas as well as macrophages related to sinuses. The esterase-staining technique could play a useful role in clinical and experimental immunology. INTRODUCTION During the past few years there has been increasing interest in defining populations of mononuclear leucocytes in human and animal peripheral blood and lymphoid organs. The original distinction was made between lymphocytes and cells ofthe mononuclear phagocyte lineage (monocytes and macrophages). Later, T and B lymphocytes were defined (Greaves, Owen & Raff, 1973) and it has become apparent that subpopulations of each of these exist. Evidence of a third population, morphologically indistinguishable from lymphocytes, has accumulated (Fr0land & Natvig, 1973; Horwitz & Lobo, 1975). These have been named L lymphocytes because of labile IgG determinants bound on their surface by highly avid Fc receptors. Currently used methods for characterizing these leucocyte populations, such as rosetting and membrane immunofluorescence, are laborious and time-consuming. There is a real need for a simple procedure to define leucocyte populations for clinical purposes. Histochemical demonstration of nonspecific esterase promises to meet this need. For some time histochemical reactions for esterase, using as the substrate oc-naphthyl acetate, has been employed for the identification of mononuclear phagocytes (Schmalzl & Braunsteiner, 1968; Yam, Li & Crosby, 1971). More recently Mueller et al. (1975) have reported localized granules of esterase reaction product in mouse lymph node T lymphocytes, whereas cells of the B-lymphocyte lineage were Correspondence: Dr D. A. Horwitz, Division of Cell Pathology, Clinical Research Centre, Watford Road, Harrow, Middlesex.
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esterase-negative. Ranki, Totterman & Hiyry (1976) used the esterase method to distinguish T lymphocytes identified by E-rosetting from B lymphocytes identified by rosetting with Staphlococcus aureus strain Cowan 1. These workers did not mention additional populations, such as L cells, nor did they comment on the usefulness of the esterase-staining method for identifying cells of the monocyte lineage, some of which are not phagocytic. No studies have been reported on human lymphoid organs. Because of the potential usefulness of the esterase method, we have undertaken systematic studies of human peripheral blood smears, purified mononuclear cell populations and sections of human lymph nodes. Techniques have been modified to ascertain the optimal conditions for differentiating between different populations of leucocytes. Moreover, the staining patterns of unstimulated lymphocytes in short-term cultures and cells exposed to T- and B-lymphocyte mitogens has been investigated. MATERIALS AND METHODS Reagents. Solutions for fixation and preparation of tissue sections. (a) Buffered formalin-acetone, pH 6-6: 20 mg Na2HPO4 was mixed with 100 mg KH2PO4, 30 ml H20, 45 ml acetone and 25 ml of 40% formaldehyde (Yam, Li & Crosby, 1971). (b) 10% neutral formol-calcium: 10 ml 40% formaldehyde was added to 1 1 mg anhydrous calcium chloride, 90 ml distilled water and adjusted to pH 7 0 with 1 N NaOH. (c) Buffered glutaraldehyde: 0-1 M phosphate buffer, pH 7-4, was added to glutaraldehyde to give a 2.5% solution. (d) Holt's gum sucrose 0-88 M: 30 g sucrose and 1 g gum acacia were mixed in the dry state and dissolved in distilled water to give a final volume of 100 ml (Pearse, 1968). (e) Gelatine-formaldehyde: 50 ml 1% gelatine was added to 50 ml 2% formaldehyde. Esterase staining. (a) Pararosanilin stock solution: 2 g pararosanilin hydrochloride (Hopkin & Williams, Chadwell Heath, Essex, U.K.) and 50 ml 2 N hydrochloric acid were heated gently, cooled to room temperature and filtered (Davis & Ornstein, 1959). (b) Incubation mixture, pH 6-1: the esterase substrate should be prepared immediately before use by dissolving 4 mg a-naphthyl acetate (Sigma Chemical Company, London) in 0 5 ml ethylene glycol monomethyl ether (Koch-Light Laboratories, Colnbrook, Buckinghamshire, U.K.) and mixed with 8-9 ml M/15 phosphate buffer, pH 7-6, and 0-6 ml hexazotized pararosanilin. The solution should not require filtration. (c) Hexazotized pararosanilin: 0 3 ml pararosanilin stock solution was mixed with 0-3 ml of freshly made 4% sodium nitrite and allowed to stand for 1 min. (d) Methyl green counterstain: 2 g methyl green was dissolved in 100 ml of heated distilled water. Chloroform-soluble material was extracted and the solution heated to 60'C to evaporate residual chloroform (Bancroft, 1975). (e) Polyclonal mitogens: phytohaemagglutinin M (PHA; Difco Laboratories, Detroit, Michigan), concanavalin A (Con A; Pharmacia, Uppsala, Sweden), and pokeweed mitogen (PWM; Grand Island Biological Co., Grand Island, N.Y.).
Proceedures. Blood and mononuclear cell smear staining. Blood was collected from each donor by venepuncture and a drop smeared onto a glass slide for esterase-staining. The remainder was heparinized (50 u/ml), diluted 1: 3, layered on FicollTriosil and centrifuged at 400 g at room temperature for 35 min. The mononuclear cells were harvested, washed three times, adjusted to 106/ml and 3 to 6 drops were pelleted onto 22 x 22 mm cover slips in a Shandon-Elliot cytocentrifuge (Shandon Scientific Company, London). The cover slips were air-dried and immediately fixed in 4VC buffered formalin-acetone for 30 sec, washed in three baths of distilled water and air-dried for at least 30 min. Both the blood smears and cytocentrifuge cover slips were stained in freshly prepared unfiltered incubation medium at room temperature for 45 min unless stated otherwise. They were rinsed free of visible deposits which sometimes formed during staining. The smears were counterstained in 2% methyl green for 5 min, rinsed in three changes of distilled water and air-dried. They were then rinsed in xylene and mounted with Ralmount (Raymond A. Lamb, London). Cell cultures. 5x 106 mononuclear cells were suspended in 5 ml RPMI 1640 containing 15% bovine serum, 2 mm glutamine and 50 pg/ml gentamycin (Flow laboratories, Irvine, Scotland). They were cultured in 30 ml tissue-culture flasks (NUNC A/S Roskilde, Denmark) for 4, 24, 48 and 72 hr. 0 5 ml (1:10) phytohaemagglutinin, 5 pg/ml concanavalin A and 0 5 ml pokeweed mitogen were added to some cultures. Five drops of polystyrene particles (Bacto Latex 0-81 pm, Difco, Detroit, Michigan) diluted 1:10 were added for the last 4 hr of culture. The cells were washed twice, an aliquot counted with a Coulter counter, viability estimated by trypan blue exclusion and cytocentrifuge smears were prepared. Tissue sections. Human lymph node specimens were fixed overnight in 10% 4VC neutral formol calcium and transferred to Holt's gum sucrose for at least 24 hr at 4VC. They were then washed in running water for 10 min, frozen in liquid nitrogen and 9 pm sections cut with a cryostat. The sections were mounted on cover slips pretreated with gelatine-formaldehyde (Pearse, 1968), allowed to dry for 1 hr at room temperature and incubated with the esterase reaction mixture for 40 min. After washing and counterstaining with methyl green, the sections were washed, dehydrated with graded alcohols, rinsed in xylene and mounted in Ralmount.
RESULTS Technical considerations Blood smears and cytocentrifuge smears were fixed and stained at various times after they were prepared. Blood smears could be stored for 2 to 3 weeks without loss of staining properties. Enzyme
gSj~ ~ '.|i ',:_. Mononuclear leucocyte populations
291
activity appeared to decrease, however, in cytocentrifuge smears of mononuclear cells. Best results were obtained when the mononuclear cells were fixed and stained shortly after the smears were prepared. Several procedures for cell fixation were compared to establish which was most satisfactory. Fixation with cold formalin-acetone for 30 sec resulted in good preservation of cell morphology and optimal esterase staining. Fixation in cold formal-calcium for 10 min (Mueller et al., 1975) also resulted in good esterase staining, but with considerable loss of morphological detail. Fixation in buffered glutaraldehyde at 4VC for 10 min (Mueller et al., 1976) resulted in loss of both esterase reactivity and morphological detail. Cold formalin-acetone is, therefore, the procedure of choice. Cytocentrifuge smears were stained for various times. Large mononuclear cells with the morphological features of monocytes were faintly positive at 20 min, and by 45 min were diffusely stained with reddishbrown reaction product. After 20 min 30 to 40% of lymphocytes developed one to four discrete granules of reaction product. After 45 min, 50 to 70% of lymphocytes had developed these punctate granules, usually close to the plasma membrane. At 90 min, the proportion of lymphocytes with discrete granules remained constant, but other lymphocytes developed ill-defined areas of reaction product (smudges) within the cytoplasm. After 3 to 5 hr of incubation with substrate, it became difficult to distinguish positive from negative lymphocytes, and all cells acquired a 'scorched' appearance. Since positive and negative cells could be distinguished optimally after 45 min of incubation, this time was selected for further studies. Examples of positive monocytes, together with positive and negative lymphocytes, in cytocentrifuge and blood smears are shown in Fig. 1. !~~~~l
- r Ii B ::i_*l fs
FIG. 1. Esterase-staining patterns in blood smears and cytocentrifuge mononuclear preparations. (a) Illustrates a diffusely staining monocyte, a lymphocyte with a discrete granule and negative lymphocytes; (b) shows a lymphocyte with one granule and a negative polymorphonuclear leucocyte; and (c) (cytocentrifuge smear) shows a diffusely positive monocyte which has phagocytosed latex particles, also several lymphocytes with one or more sharply defined granules of reaction product and several negative lymphocytes. (Magnification x 540.)
Staining patterns oflymphocytes Counts of granular and diffuse esterase-positive and of esterase-negative cells made in cytocentrifuge preparations of peripheral blood mononuclear cells from healthy subjects are shown in Table 1. The diffusely stained cells, which were of the mononuclear phagocyte lineage (see below), were subtracted from the total cell counts in order to determine the proportion's of lymphocytes with granular esterasestaining (Table 2). These results were compared with the percentage of E rosette-forming T cells in the same mononuclear suspension and the percentage of granular-staining lymphocytes in blood smears. Table 2 reveals that the great majority of blood lymphocytes were positive for both E-rosetting and granular-staining. The percentages of granular-staining cells in blood smears and cytocentrifuge preparations were similar. Although in some subjects the percentage of granular-staining cells approached that of E rosette-forming cells, in others the number of esterase-positive cells was 10 to 20% lower. Evidently most, but not all, T lymphocytes give a localized esterase reaction product. The correspondence between these two populations was confirmed by separating E-rosetting cells from the whole population. 80 to 90% of T lymphocytes, purified as described above and cytocentrifuged onto cover slips, showed the characteristic esterase-staining (Fig. 2b). Less than 8% of lymphocytes in B and L preparations showed granular staining, and this was due to residual T lymphocyte contamination (Fig. 2a). These preparations contained 6 to 8% E-rosetting lymphocytes.
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TABLE 1. Esterase-staining of mononuclear cells from normal
subjects Positive (%) Granular pattern
Subject
61 62 67 52 66 52 61 62 69 66 61-8
J.D. D.H. J.W. J.C. H.E. A.K. L.B. (Initial) (Repeat)* A.T. (Initial) (Repeat)* Mean *
Diffuse pattern 19 11 13 35 16 28 19 10 20 21 19-2
Negative (0) 20 27 20 14 19 20 20 28 11 13
19'2
Repeat examination 3 weeks after the initial study.
TABLE 2. Comparison of T lymphocytes detected by the E-rosette method and esterase technique
Granular-staining lymphocytes (0) Subject J.D. J.W. A.T. L.B. A.K. A.N. T.A. H.E. J.E. Mean
E rosettes (0)
86 84 82 81 78 77 75 73 67 78-1
Blood smear 69 76 81 70 72 65 63 77 65 70 9
Mononuclear cells* 72 77 82 68 72 64 70 75 59 71-0
* Cytocentrifuge preparations of mononuclear cells obtained by Ficoll-Triosil density gradient centrifugation.
Mononuclear lymphocytes prepared by Ficoll-Triosil centrifugation were cultured for 72 hr and developed changes in esterase-staining patterns. The proportion of granular-staining lymphocytes declined during the first 2 days in culture, but thereafter increased (Fig. 3). Other lymphocytes were stimulated with PHA, Con A and PWM, and esterase-staining determined after 24, 48 and 72 hr of culture. These polyclonal mitogens did not alter the esterase activity in small lymphocytes, but the activity declined as lymphocytes enlarged and became immature blasts. Most transformed cells showed little or no staining (Fig. 4). In some cells clusters of very fine granules were found.
Staining patterns of mononuclear phagocytes A major advantage of the esterase technique is that it identifies cells of the mononuclear phagocyte lineage at a relatively early stage of differentiation. 19% of blood mononuclear cells showed diffuse esterase-staining. Approximately 20% of these cells in all normal subjects tested did not phagocytose
Mononuclear leucocyte populations
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FIG. 2. Examples of mononuclear populations stained for esterase. (a) Negative non-T cells separated by Ficoll-Triosil centrifugation of E rosette-forming cells; (b) positive T cells after red cells have been lysed by ammonium chloride. (c to f) Mononuclear phagocytes after short-term culture; (c) 4 hr, examples of diffusely stained cells, one of which has ingested latex particles while the other has not; (d) 24 hr, almost all diffusely positive cells are phagocytic; (e) 48 hr, shows the development of a subpopulation with a round, eccentrically placed nucleus and cytoplasm with increased intensity of staining; phagocytic capacity is reduced; and (f) 72 hr, two non-phagocytic cells, one of which has developed numerous secretory granules; these are easily distinguishable from the smaller, more sharply defined latex particles. (Magnification x 540.) 80
r
60 0
0'< c
.0 71i
40
20
0
I
4
24
48
72
Time (hr)
FIG. 3. Granular esterase-staining of mononuclear cells prepared by Ficoll-Tricsil density gradient centrifugation. Error bars denote s.e.m.
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FIG. 4. Lack of esterase-staining of lymphocytes transformed by concanavalin A after 72 hr of culture. Identical fields by bright field (a) and phase-contrast (b) microscopy. Two large immature blast-like cells are shown which are esterase-negative; a positive small lymphocyte and a phagocytic monocyte are included for comparison. (Magnification x 540.)
latex particles in dilute suspensions. Examples of these cells are shown in Fig. 2c. When the cells were cultured for 24 hr. more than 95% of the diffuse esterase-positive cells were phagocytic in each of five subjects (Fig. 2d). Another notable feature was that the total number of esterase-positive cells increased during the initial 48 hr of culture. This remains true even when corrections are made for non-viability (Fig. 5). The increase cannot be explained by cell multiplication because less than 02%/o of unstimulated 30 r-
01
20
M
0'
C3
(-)
10
0
4
48
24
72
Time (hr)
FIG. 5. Percentage and phagocytic capacity of diffuse esterase-stained mononuclear cells after short-term culture. (0) Indicate the proportion of diffusely positive cells and (o) indicate diffusely positive cells that have phagocytosed latex particles. The mean numbers of trypan blue viable cells from 4 to 74 hr were > 99Y%, 95%4, 85%4 and 78Y% respectively. Data points have been adjusted downwards to account for dead cells and therefore indicate an increase in the absolute number of diffusely positive cells.
ti. ~ ~W.;
295
Mononuclear leucocyte populations
mononuclear cells in culture divide (Horwitz, 1972). Therefore, this observation implies maturation of precursor cells morphologically indistinguishable from lymphocytes into esterase-positive monocytes. At 48 hr and subsequently, the proportion of phagocytic diffusely stained cells declined and changes in monocyte morphology were evident. At the start of the culture period the cells were round and the nuclei had a typical kidney-shaped, indented or lobed appearance (Fig. 2c). After 48 hr of culture the intensity of esterase-staining had increased and many cells acquired an oval shape. The nuclei had likewise become oval and eccentrically placed in the cell (Fig. 2e and f). Some of the cells developed numerous refractile secretory granules (Fig. 2f). Possibly these changes represent a stage of differentiation towards a non-phagocytic cell, such as an epithelioid cell. Staining patterns of lymph nodes Sections of fixed lymph nodes showed well-defined areas of esterase reaction product. Intense diffuse staining was observed in macrophages lining the marginal sinus and cortical sinuses connecting with the more concentrated network of sinuses in the medulla (Fig. 6). T lymphocytes with granular esterasestaining were easily identifiable under the high power (magnification x 40). At low power, areas of esterase reaction product less intense than that lining the sinuses appeared as a distinct reddish blush in the section. These corresponded to the paracortical (T-dependent) areas of the nodes.
_
~~~~4
*|
:-'b4i
X
~~~~~~~~~~~~~~~i
FIG. 6. Esterase-staining of lymph node. (a) Lymphoid modules (low-power magnification), heavily stained macrophages line marginal sinuses and paracortical areas contain 'blush' of stain. (Magnification x 325.) (b) High-power magnification, many paracortical T cells with discrete granules surround area of esterase-negative B cells. (Magnification x 540.)
Clinical examples Two examples of the clinical usefulness of esterase-staining are shown in Table 3. The first is the rapid detection of increased numbers of cells of the mononuclear phagocyte lineage in patient G.A. More than one-half of his mononuclear cells showed diffusely positive staining. Some of these were small round cells which morphologically resembled lymphocytes. After overnight culture, however, 97% of the diffusely positive cells had phagocytosed latex particles. Secondly, in both cases of immunodeficiency syndromes there was marked discrepancy in the number of E rosette-positive cells and lymphocytes with granular esterase-staining. In patient C.C. the number of esterase-positive lymphocytes was less than 20% of the number of E-rosetting cells. These findings suggest that immature T cells lack esterase activity.
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TABLE 3. Examples of abnormalities detected by esterase staining T lymphocytes (%)t
Granular Monocytes (%)* E rosette-forming esterase-staining
Comments
Patient
Diagnosis
G.A.
Congenital hypogammaglobulinaemia
62
69
50
Marked increase in the percentage of blood monocytes. Only 72% of T cells were esterase-positive. More than 80% of T cells in normals were esterase-
C.C.
Congenital hypogammaglobulinaemia
29
67
11
Marked decrease in esterase-positive T cells. Only 16% were positive
positive
* These are diffuse esterase-staining, expressed as percentage monocyte population.
t Expressed as percentage of total lymphocytes.
DISCUSSION Previous investigators had shown that esterase-staining could be used to identify either monocytes (Schmalzl & Braunsteiner, 1968) or a subclass of lymphocytes with a granular reaction product (putative T lymphocytes) (Mueller et al., 1975). The main purpose of this investigation was to develop a single method for the simultaneous demonstration of esterase-positive and -negative lymphocytes, and phagocytic or precursor cells of the mononuclear phagocyte lineage. For that purpose different methods of preparing, fixing and staining the cells have been compared. Both preparations from peripheral blood smears and cytocentrifuged Ficoll-Triosil mononuclear cells gave good staining. Three major categories of cells were readily identified, and counts made on the two preparations from the same individual showed excellent correspondence. There was no measurable loss of any cell population identifiable by the esterase technique during the preparation of mononuclear cells by density gradient centrifugation. Brown & Greaves (1974) reported that density gradient centrifugation decreased the percentage of E rosette-forming cells. Each preparation has advantages. The blood smear uses a small drop of blood which is dried in the conventional way and requires no special processing. Even the reagents for staining are commercially available (Tucker, Pierre & Jordan, 1977). Hence blood films can be used in looking for gross deficiencies. However, performing differential counts of enough cells to obtain statistically meaningful results is laborious and time-consuming. Density gradient preparations are widely used in clinical immunology laboratories, and it seems likely that this will become the routine method for preparing cells for esterase-staining, because of the high quality of the cells and the speed with which differential counts can be made. The great majority of blood lymphocytes were both E-rosetting and esterase-positive. The proportion of esterase-positive lymphocytes was usually less than that of E rosette-forming cells. Several explanations can be suggested. One possibility is a threshold effect on staining, that a proportion of T lymphocytes is not stained with the standard conditions used. However, this is unlikely because longer incubation results in weak reactions which may occur in all lymphocytes. A second possibility is that esterasenegative lymphocytes are the same as the subpopulation of T lymphocytes with Fc receptors. The Fc receptor-bearing subpopulation can comprise up to 20% of T cells) Ferrarini et al., 1975). This possiblity has not been excluded, but is unlikely. A third possibility is that esterase-negative E-rosetting cells are immature T lymphocytes. Consistent with this interpretation is the observation that mouse cortical thymocytes are nearly all esterase-negative (Mueller et al., 1975). Moreover, when T lymphocytes
Mononuclear leucocyte populations
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were transformed into immature blasts by polyclonal mitogens in culture, loss of esterase activity was observed. For these reasons, the latter explanation seems most likely and could have clinical application in revealing defects in T-lymphocyte maturation. This is illustrated by the two patients with immunodeficiency syndromes described in Table 3. The esterase technique also provides useful information about cells of the mononuclear phagocvte lineage. In addition to the well-known fact that monocytes are esterase-positive, it is possible to obtain information about their state of maturity. Approximately 20% of diffusely esterase-stained cells in peripheral blood from normal subjects were not phagocytic. When the cells were cultured for 24 to 48 hr the absolute number of esterase-stained cells increased, and 98% of these were phagocytic. These results suggest maturation of esterase-negative cells into cells with diffuse esterase-staining and the maturation of phagocytic capacity. After 72 hr in culture the proportion of phagocytic cells declined and of oval cells with the distinctive appearance of epithelioid cells increased. The cytoplasm of some of these cells was filled with secretory granules. Thus, different subpopulations of mononuclear phagocytes can be identified with esterase-staining. The present observations suggest that the proportion of monocyte-like cells in human blood is higher than generally supposed. This proportion may be further increased in patients with some infections, inflammatory diseases and malignant states. For example, in patients with systemic lupus erythematosus 40 to 50% of mononuclear cells are blood monocytes, as shown by acridine orange vital fluorescence, and this finding explains, at least in part, the decreased mitogenic reactivity (Horwitz & Garrett, 1977). Moreover, patients with systemic lupus erythematosus, other connective tissue disorders and infections also have increased numbers of monocyte precursors incorporating thymidine into DNA (Horwitz, 1972). In general, the esterase technique can provide useful information, not only about the total number of monocytes and T lymphocytes in the circulation, but also their degree of maturity. Esterase-negative cells include granulocytes (Yam et al., 1971), B lymphocytes and lymphocytes with high-affinity Fc receptors for IgG, termed L lymphocytes (Horwitz & Lobo, 1975). Even these may show weak reaction products after prolonged incubation with substrate. Besides being applicable to cell suspensions, the esterase technique can be used on sections of lymph nodes and other tissues. It provides a simple and reliable technique for demonstrating diffusely stained macrophages and granular-staining mature T lymphocytes. These results appear be to applicable to humans and several species of experimental and domestic animals. The identification of these cells without the need for specific antisera and without the uncertainties of in situ resetting is a considerable technical advance. It seems likely that the esterase technique will be applied routinely in many clinical and experimental laboratories. We are indebted to Dr D. Webster and Dr G. Slavin for providing blood samples and lymph node specimens. This work was supported by the John A. Hartford Foundation grant 74466, and United States Public Health Service grant 11766. Dr Horwitz is a recipient of the Clinical Scholar award from the Arthritis Foundation. REFERENCES
BANCROFT, J.D. (1975) Histochemical Techniques, 2nd edn, p. 260 and p. 322. Butterworths, London. BROWN, G. & GuAvEs, M.F. (1974) Enumeration of absolute numbers of T and B lymphocytes in human blood. Scand. 5. Immunol. 3, 161. DAVIS, B.J. & ORNSTEIN, L. (1959) High resolution enzyme localization with a new diazo reagent 'hexazonium pararosaniline'. 5. Histochem. Cytochem. 7, 297. FERRARINI, M., MORETTA, L., ABRILE, R. & DURANTE, M.L. (1975) Receptors for IgG molecules on human lymphocytes forming spontaneous rosettes with sheep red cells. Europ. 5. Immunol. 5, 70. FRoLAND, G.F. & NATVIG, J.B. (1973) Identification of three different lymphocyte populations by surface markers. Transplant. Rev. 16, 114.
GREAvEs, M.F., OWEN, J.J.T. & RAFF, M. (1973) T and B Lymphocyte Origins, Properties and Role in Immune Responses. Excerpta Medica, Amsterdam. HORWITZ, D.A. (1972) The development of macrophages from large lymphocyte-like cells in the blood of patients with inflammatory diseases. J. clin. Invest. 51, 760. HORWITZ, D.A. & GARRETT, M.A. (1977) Mitogenic reactivity of human lymphocytes. I. Decreased responsiveness in systemic lupus erythematosus, rheumatoid arthritis and scleroderma: technical considerations. Clin. exp. Immunol. 27, 100. HORWITZ, D.A., & LOBO, P.I. (1975) Characterization of two populations of human lymphocyte-bearing immunoglobulins. J. clin. Invest. 56, 1464. MUELLER, J., BRUN DEL RE, G., BUERKI, H., KELLER, H.U.,
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HESS, M.W. & COTTIER, H. (1975) Nonspecific acid esterase activity: a criterion for differentiation of T and B lymphocytes in mouse lymph nodes. Europ. 5. Immunol. 5, 270. MUELLER, J., KELLER, H.U., BRUN DEL RE, G., BUERKI, H. & HESS, M1.W. (1976) Nonspecific esterase activity in T cells. Immune Reactivity of Lymphocytes (ed. M. Feldman and A. Globerson), p. 117. Plenum Publishing Corporation, New York. PEARSE, A.G.E. (1968) Histochemisiry, Theoretical and Applied, 3rd edn, Vol. 1., p. 602 and p. 728. Churchill, London. RANKI, A., TOTTERMAN, T.H. & HAYRY, P. (1976) Identi-
fication of resting human T and B lymphocytes by acid anaphthyl acetate esterase staining combined with rosette formation with Staphylococcus aureus strain Cowan I. Scand. 7. Immunol. 5, 1129. SCHMALZL, F. & BRAUNSTEINER, H. (1968) Cytochemische Darstellung von Esterasere-aktivitat in Blut - und Kncckenmarkszellen. Kim'. Wschr. 46, 642. TUCKER, S.B., PIERRE, R.V. & JORDAN, R.E. (1977) Rapid identification of monocytes in a mixed mononuclear cell preparation. 3. immunzol. Methods, 14, 267. YAM, L.T., Li, CY. & CROSBY, W.H. (1971) Cytochemical identification of monocytes and granulocytes. Am. 7. Clin. Path. 55, 283.
