Origins and maturation patterns of azurocytes in the meadow vole (Microtus pennsylvanicus)

Camp. Biochem.Physiol.Vol. 99C, No. l/2, pp. 219-230,1991 0

Printed in Great Britain

ORIGINS AND MATURATION AZUROCYTES IN THE MEADOW

0306-4492/91 $3.00 + 0.00 1991 Pergamon Press plc

PATTERNS OF VOLE (MICROTUS

PENNSYLVANICUS) STEVEMIHOK* and BILL SCHWARTZ Environmental

Research Branch, Whiteshell Research Establishment, Pinawa, Manitoba, Canada ROE 1LO (Received 23 August 1990)

Abstract-l. The origins, maturation sequence, and traffic patterns of azurocytes (AZ) were studied in viuo in the meadow vole (Microtus pennsyluanicus) with the aid of various biological response modifiers. 2. AZ originated from lymphocyte-like precursors in the bone marrow. These precursors entered into intense mitotic activity in the bone marrow following progestin treatment. 3. While still in the bone marrow, AZ precursors differentiated into granulated cells with some of the histochemical properties of AZ. 4. At this time, maturation could be blocked completely with cyclophosphamide. Hydroxyurea and cyclosporin A inhibited maturation significantly, but did not block it. 5. Estradiol, hydrocortisone, prostaglandin E,, and conditioned medium had no effect on the numbers of cells induced. 6. Transitional stages between a granulated bone marrow precursor and an AZ were rarely observed, suggesting rapid synthesis of AZ inclusions upon terminal differentiation. 7. After release from the bone marrow, AZ were common in the blood only. Modest numbers of cells were also found in the red pulp of the spleen, and the thymic cortex. 8. AZ in the thymus were sometimes found in clusters, and were often associated with septa. Very low numbers of mature AZ were detected in the bone marrow. 9. AZ were absent from lymph nodes and Peyers patches, and were never observed in the B-lymphocyte areas of the spleen. IO. Based on experiments with dextran sulphate, AZ and their precursors never entered the recirculating lymphocyte population. AZ in the blood or tissues were never observed in mitosis, and were never observed degranulating. 1 I. Cyclophosphamide or hydrocortisone had no effect on AZ numbers when administered at the peak of their occurrence in the blood. 12. Although no clear function of the AZ was elucidated, experiments confirmed a high degree of similarity between its properties and those of cytotoxic cells found in other mammals.

INTRODUCTION The incredible diversity of lymphocyte types and functions is masked by the nondescript appearance of cells on Romanowsky-stained smears. In most instances, variation in morphology among individuals, and among species, reveals little about a cell’s activities. An important exception to this observation is the association of granulated lymphocytes with cytotoxic activity (Young and Cohn, 1986; Herberman et al., 1986). In particular, large granular lymphocytes (LGL) have been associated specifically with natural killer (NK) activity. These cells are characterized by the presence of azurophilic cytoplasmic granules, a reniform nucleus, a clear cytoplasm, and a low buoyant density (Ortaldo and Herberman, 1984). In rats, mice, and humans, virtually all of the NK activity in peripheral blood lymphocytes has been attributed to LGL. A recent study in rats has also identified LGL with the induction of lymphokine*Please address all correspondence

to Dr Steve Mihok at: Tsetse Research Program, International Centre of Insect Physiology and Ecology, P.O. Box 30772, Nairobi, Kenya (Telephone: 802509, Ext 255; FAX: 803360).

activated killer cell activity under the influence of high levels of interleukin-2 (Sarneva er al., 1989). Other lymphoid cells from mammals also contain granules or inclusions, but these cells have not been investigated as thoroughly as LGL. Some of these cells are not normally found in blood: e.g. the granulated intraepithelial lymphocyte from the small intestine (Huntley et al., 1984), and the granulated metrial gland cell from the pregnant uterus (Bulmer et al., 1987). An unusual lymphoid cell is, however, found in the blood of guinea-pigs: the Kurloff cell (KC). The KC contains a single large inclusion, is common in females, especially during pregnancy, and can be induced by estrogen (Revel], 1977). Although possessing natural killer activity, the precise function of the KC is unknown. Similarly, the kinetics of the KC and its relationships to other cells are just now being elucidated (Sandberg and Hagelin, 1986; Landemore et al., 1987; Buat et al., 1986; Thomson et al., 1988). A recent addition to the family of unusual lymphoid cells in mammals is the vole azurocyte (AZ) (Mihok et al., 1987). This cell contains numerous large azurophilic inclusions similar to those found in LGL in other mammals. The AZ is characteristic of 219

STEVEMIHOK and BILL SCHWARTZ

220

voles of the genus Microtus, and appears to be absent from closely-related species of cricetid rodent. The cell is particularly common in late pregnancy (Mihok, 1987), and can be induced with progestins and interferon inducers (Mihok and Schwartz, 1991). Hence, it shares many features in common with the KC. The azurocyte’s function is unknown. Based on similarities with the KC and LGL, we speculated previously that it might be a NK cell unique to the vole (Mihok et al., 1987). In this paper, we report details of the basic immunobiology of the AZ from laboratory studies of the meadow vole (Microtus pennsyluanicus). Through the use of various biological response modifiers, we have been able to trace the development and traffic patterns of the cell. This work has confirmed our earlier premise that the cell most closely resembles the cytotoxic cells found in other mammals. It is hoped that this information will stimulate further studies of the vole as a new model for basic research in cellular immunology and reproductive physiology. MATERIALS AND METHODS Animals Meadow voles were obtained from a colony established in 1985 at the Whiteshell Research Establishment near Pinawa, Manitoba, Canada (Mihok and Schwartz, 1991). Animals were used in experiments at about 3-14 weeks of age. Unless noted otherwise, a minimum of six voles of matched age and sex were used in experimental treatments. Hematology Blood was collected in heparinized microhematocrit tubes through the suborbital sinus. Leukocyte counts and thin smears were collected with standard manual hematological techniques (Mihok and Schwartz, 1991). Bone marrow smears were prepared from femoral marrow and stained with Azure B-Eosin Y (ICSH, 1984). Most marrow smears were examined qualitatively only. In a few cases, a 500-cell differential count was performed. Histology and histochemistry Tissues were fixed for one day in methanol-formalinacetic acid (85: lO:S, MFAA) (Mayrhofer, 1980); they were then transferred to 70% ethanol for storage. After some initial experimentation, this fixative was found to be optimal for the simultaneous staining of AZ and other cells (Appendix). Tissues were collected from most experiments, including the work with various biological response modifiers reported in Mihok and Schwartz (1991). Tissues were processed for paraffin-embedded histology within a few weeks of collection. We always examined sections from the spleen, thymus, and mesenteric lymph nodes. Impression smears were made occasionally from these organs so that cell morphology could be examined in detail. Sections from Peyers patches, lungs, liver, kidneys, adrenals, pituitary, testes, uterus, and ovaries were prepared in a few experiments. Other organs (brain, skin, salivary glands, gastrointestinal tract, etc.) were examined in the initial work reported in Mihok and Schwartz (1991). To provide information on the tissue distribution of AZ during pregnancy, we retrieved previously-fixed material from wild pregnant voles for histology (Mihok et al., 1987). This material had been stored in an alcoholic formalinbased fixative for a few years, and hence may have lost some of its staining properties. Nevertheless, we examined these old tissues with the same histochemical stains used for freshly-collected experimental material. All tissues sections were stained with Hematoxylin-Eosin for an initial overview. Impression smears were stained with

Azure B-Eosin Y. Additional sections were made for interesting material; these were stained with: Aldehyde Fuchsin (AF), Alcian Blue pH 1.0 (AB), Periodic Acid Schiff after amylase digestion (PAS), and Alcian Blue pH 1.0 followed by Periodic Acid Schiff without amylase digestion (AB-PAS). More specific enzymatic stains were used occasionally on selected material to confirm the identity of AZ (Mihok et al., 1987). Experiments Arfifical induction of azurocytes. In all experiments, AZ were induced artifically by a single S.C. injection of 5 mg of medroxyprogesterone acetate (MPA) dissolved in 0.1 ml sesame oil. This standard protocol was adopted following initial experimentation with a variety of hormones and biological response modifiers (Mihok and Schwartz, 1991). Maturation and trafic patterns. This experiment was designed to test the effects of various immunomodulators on the blood and tissue dynamics of the AZ. In all treatments, voles were injected with MPA on day 0. The control groups received MPA only, experimental groups received other drugs before or after the administration of MPA. A baseline leukocyte and differential count was taken from each vole at the start of each treatment. The following compounds and dose levels were used: (a) cyclophosphamide (Homer)-300 mg/kg injected i.p. in distilled water; (b) hydrocortisone-21-acetate (Sigmab 1 mg/vole injected S.C. in 0.1 ml sesame oil; (c) dextran sulphate (SigmaFSO mg/kg injected i.p. in distilled water (Bradfield and Born, 1974); and (d) estradiol benzoate (Sigma)-5 mg/vole injected S.C. in 0.1 ml sesame oil. Drugs were used in various ways at four times during the maturation sequence of the AZ: (i) preinduction: in an attempt to block or prime induction, we pretreated voles on day -3 with either cyclophosphamide or estradiol. Voles were bled on days -3, 0, 3 and 7. An additional group was pretreated on day - 1 with cyclophosphamide, and was bled on days - 1, 0, 3 and 7; (ii) early maturation: prior to the appearance of AZ, we attempted to block induction on day 1 with cyclophosphamide or hydrocortisone (bled on days 0, 3, 3 + 4 hr, 4, 7 and 10) or estradiol (bled on days 0, 3 and 7); (iii) early appearance in blood: just at the first appearance of AZ, we attempted to interfere with maturation and traffic patterns. Voles were treated on day 3 with cyclophosphamide or hydrocortisone (bled on days 0, 3, 3 + 4 hr, 4, 7 and lo), or dextran sulphate (bled on days 0, 3 and 3 + 4 hr), or estradiol (bled on days 0, 3, 7 and 10); (iv) peak occurrence in blood: when AZ were at their peak in the peripheral blood on day 7, we examined the response of the cell to various drugs. Voles were treated on day 7 with cyclophosphamide or hydrocortisone (bled on days 0, 3, 7, 7 + 4 hr, 8, 10 and 15) or dextran sulphate (bled on days 0, 3, 7, 7 + 4 hr and 8). A single control group of 10 voles was used for all treatments; voles were bled on days 0, 3, 7, 10 and 15. In total, 126 voles were used in the entire suite of 13 experimental treatments. Localization of precursors. To determine the location of cells involved in the maturation of the AZ, we injected i.p. a few selected voles with 5 mg/kg colchicine (Sigma). Tissues were harvested 4 hr later and examined for mitotic figures. Voles were treated with colchicine on days 1, 2, and 3 after treatment with MPA. A few splenectomized and sham-operated voles from Mihok and Schwartz (1991) were also treated one month after receiving an implant of MPA. Tissue sections and bone marrow smears were examined for AF, AB or PAS staining in mitotic figures or immature cells. Azurophilic staining of granules was checked for correspondence to staining with AF, AB or PAS in duplicate slides. Normal haematopoietic lineages were differentiated

Azurocyte maturation in the vole Azurocytes 1.5

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Fig. 1. Absolute changes in AZ counts on day 7 in meadow voles treated with various drugs on day - 3, - 1, + 1 or + 3 relative to treatment with MPA on day 0. Asterisks denote values significantly different from controls receiving MPA only.

Fig. 3. Absolute changes in lymphocyte and neutrophil counts on day 7 in meadow voles treated with estradiol on day -3, + 1 or +3 relative to treatment with MPA on day 0. Asterisks denote values significantly different from controls receiving MPA only.

from AZ precursors on morphological criteria in conjunction with specific staining for lipids, peroxidase and anapthyl acetate esterase (Mihok et al., 1987). Immunomodulation. This experiment was designed to compliment and confirm the results of previous experiments. Drugs known to affect the proliferation or action of macrophages, T-lymphocytes, or lymphokine-activated killer cells were chosen for investigation. A mitotic inhibitor with minimal myelotoxicity was also tested to compare with cyclophosphamide. In each experimental treatment, voles received MPA on day 0 followed by other drugs. Matching control groups received only the other drugs. A single group received only MPA. Voles were bled on days 0, 3 and 7. Unlike previous experiments, most organs were processed for histology at the end of the experiment. Fresh weights of the spleen were also taken at autopsy. Drug treatments consisted of the following: (a) cyclosporin A (Sandoz)---50 mg/kg injected S.C. in 0.1 ml sesame oil on days 1 and 2 (Thomson et al., 1981); (b) PGE, (Sigma)-25 pg injected S.C.in 0.1 ml sesame oil on days 1 and 2 (Gentile et al., 1983); (c) hydroxyurea (Sigma)-1 g/kg injected i.p. in PBS at hour 24 and hour 31 (Hodgson et al., 1975); (d) conditioned medium-voles were injected S.C. with a 1 ml solution of 15% gelatin and conditioned medium from an interleukin-2 secreting cell line, at hours 24, 31, 48 and 55 (Chang et al., 1984). The source culture was MLA 144, a T-cell lymphoma obtained from a gibbon

ape (Yang ef al., 1986). Conditioned medium was harvested after a IO-fold increase in cell concentration during four days of cell multiplication from an initial concentration of 10’ cells/ml.

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Statistical analyses

As all voles were bled prior to any experimental manipulation, results have been analyzed with repeated measures analysis of variance. Sample statistics are reported as the mean f standard error. RESULTS

Maturation

Induction

and trafic

patterns

of AZ was nearly completely blocked by

administration of cyclophosphamide either one or three days after the injection of MPA (Fig. 1). In contrast, administration of cyclophosphamide prior to MPA had an insignificant effect on AZ. All other attempts to affect AZ induction with the use of hydrocortisone or estradiol, either before or after injection of MPA, were unsuccessful (Fig. 1). Similarly, treatment of voles with cyclophosphamide, hydrocortisone, or dextran sulphate on day 7 did not affect AZ significantly during the second week of the experiment (Fig. 2). AZ reamined stable from day 7 through 10 in all groups, declining only on day 15. In sharp contrast to the insensitivity of AZ to various drugs, other leukocytes reacted significantly to all drug treatments. Dextran sulphate administered on day 3 or day 7 resulted in a 7-fold increase in lymphocytes, and a 17-fold increase in neurtrophils, 4 hr after treatment. Estradiol adminstered at any time relative to MPA resulted in a 2- to 3-fold increase in neutrophils by day 7 (Fig. 3). Lymphocytes either decreased (estradiol day + 1 or +3) or increased (estradiol day - 3) when voles were treated with e&radio1 and MPA (Fig. 3). Lymphocytes and neutrophils were highly sensitive to hydrocortisone and cyclophosphamide throughout the period of AZ induction. Treatment with cyclophosphamide resulted in an immediate decrease in lymphocytes that was sustained in following days (Fig. 4). The decrease was particularly pronounced in voles treated with cyclophosphamide on day 7. Combined with the lymphopaenia induced by MPA alone

STEVEMIHOK and BILL SCHWARTZ

222 Cyclophosphamide Day 1

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Fig. 4. Absolute changes in lymphocyte and neutrophil counts in meadow voles treated with MPA on day 0, followed

by treatment

with cyclophosphamide or hydrocortisone on days + 1, + 3, or + 7. Asterisks denote values significantly different from zero.

(Mihok and Schwartz, 1991), this additional treatment resulted in lymphocyte counts as low as 1 x lo9 cells/l through to day 15. Similar immediate and prolonged decreases in lymphocytes were effected through treatment with hydrocortisone (Fig. 4). However, the magnitude of these changes was less than with cyclophosphamide. Neutrophils reacted differently to the two drugs. Cyclophosphamide induced a transient rise at 4 hr, followed by a return to normal at 24 hr (Fig. 4). Neutrophils decreased to very low numbers in the next few days, and then rebounded to 2-3 x normal values as the bone marrow recovered. In contrast, hydrocortisone induced a transient rise in neutrophils limited to the first 24 hr (Fig. 4). Localization of precursors

In voles treated with colchicine after MPA, levels of mitotic activity in the spleen, thymus, mesenteric lymph nodes, and Peyers patches were low, and were quite variable among individuals. In contrast, levels of mitotic activity in the bone marrow were high, and were clearly different from controls. Smears from these voles contained an average of 13-21% mitotic figures (N = 18), compared with 1.6% in colchicine-treated controls (N = 4), and 0.9% in untreated controls (N = 16). On day 1, mitotic activity was limited almost exclusively to small-to-medium-sized cells with scanty cytoplasm resembling lymphocytes (Fig. 5A). A small number of

these dividing cells contained azurophilic granules on day 2, with more showing granulation on day 3 (Fig. 5B). The granules stained intensely with AF, but were either negative, or stained weakly with PAS; no staining activity was observed with ANAE (LXnapthyl-acetate esterase). The granules were larger, rounder and with a more distinct outline than those observed in promyelocytes (Fig. SC); they also did not stain for myeloid markers such as peroxidase and lipids. AZ were not observed in the bone marrow of control voles (N = 16). They were, however, present in very low numbers in MPA-treated voles (maximum 0.6%). Lymphocytes represented 5-27% (mean 12%) of the bone marrow cells in controls. Mitotic cells in colchicine and MPA-treated voles resembled lymphocytes, and were about as abundant as lymphocytes were. Although lymphocyte-like cells were clearly proliferating after MPA treatment, we could not detect these changes in bone marrow differentials without the use of colchicine (N = 103) from various experiments. We also never observed clearly elevated numbers of blast cells following MPA treatment. Possible AZ blast cells were never numerous enough to identify unequivocally (Fig. 5A), and could easily have been blast cells of other lineages. In contrast, it was quite easy to detect pronounced changes in blast cell numbers in bone marrow differentials during myeloid cell proliferation following cyclophosphamide treatment (Fig. 5D).

Azurocyte maturation in the vole When AZ appeared in the peripheral blood on days 3 or 4, they resembled “normal” AZ observed in wild voles. Staining reactions for AF and PAS were stable throughout the period that AZ were monitored. Transitional stages from a granulated lymphocyte in the bone marrow to an AZ in the blood were rarely observed. Similarly, we never observed a mature AZ in mitosis. The presence of AZ in tissues was only crudely related to the levels observed in the blood. In most cases, AZ could not be found in tissue sections from controls. AZ were present in tissues from MPAtreated and pregnant voles, but not in a consistent pattern. Altogether, we examined hundreds of tissue sections from days 1-4, 7-10 and 15 after MPA treatment, without discerning a clear traffic pattern for the cell. In tissues with a rich blood supply, such as the spleen, lungs and placenta, scattered AZ were detected with specific staining of the inclusions (AF, PAS or AB-PAS, see Appendix). Locating AZ with general stains such as H&E was difficult. AZ were present in the placental labyrinth during pregnancy, but were too rare for us to note any relationship with the stage of pregnancy. In the spleen, AZ were found mostly in the chords and sinuses of the red pulp, usually as single cells with no obvious association with other cells. AZ were never found in the white pulp, but were occasionally present in the marginal zones. AZ never exceeded about 20 per 1000 x field in the red pulp, and were then often as few as l-3 per field. In the lungs, AZ were present in large numbers on day 7. Their presence was, however, associated with a pronounced interstitial leukocytic infiltration of the lungs. This may very well have been a sideeffect of the use of MPA. AZ were conspicuously absent from lymph nodes and Peyers patches. In contrast, AZ were routinely found in the thymus in MPA-treated voles, and occasionally, in controls. AZ appeared in small numbers two days after treatment with MPA; their numbers appeared to increase in the second week after treatment. Cells were found mainly along the outer edges of the cortex (maximum of 20 per 1000 x field, typically 4-8) or near septa (Fig. 5E). AZ were also sometimes present in the medulla, but were fewer in number (maximum of 5 per 1000 x field), and were mostly located near the cortico-medullary junction. AZ in the thymus often occurred in clusters of 2-4 cells, but we could discern no particular relationship with surrounding cells. Transitional forms similar to the granulated lymphocytes observed in the bone marrow were observed occasionally. As fine AFf granules were difficult to detect in paraffin-embedded sections, we may have missed these cells in the majority of our histological material. Induction of the AZ was not associated with histological changes in the adrenals. MPA induced endometrial gland development in the uterus, but did not result in recruitment of AZ. Pituitaries of day 7 MPA-treated voles contained massive numbers of PAS+ cells (gonadotropes?), that were present in small numbers only in controls, and in day 3 MPAtreated voles. AZ induction with MPA was associated with the depletion of lymphocytes from lymphoid organs.

223

Hence, lymphopaenia resulting from MPA treatment (Mihok and Schwartz, 1991) probably reflected a decrease in lymphocyte production, as opposed to a redistribution of lymphocytes. During the first few days following MPA treatment, lymphocyte numbers were reduced in the red pulp of the spleen (Fig. 5F), the medulla of the mesenteric lymph nodes and thymus, and, to a lesser extent, the cortex of the nodes and thymus. Germinal centres of the lymph nodes and spleen remained relatively unaffected in both size and number. In the thymus, transient degenerative changes also occurred. Three days after MPA treatment, normal thymic architecture was disrupted, many pyknotic lymphocytes were observed, and there was a proliferation or recruitment of large, lightly-staining, PAS+, AFcells, presumably macrophages (Fig. 5G). Many of these cells contained ingested lymphocytes. Normal thymic structure was restored by day 7, although some lymphoid depletion was still evident. Similarly, near normal numbers of lymphocytes were present in the spleen on day 7. In the mesenteric lymph nodes, recovery of normal lymphocyte numbers was slow. On day 7, relatively few cells were present in the medullary chords and sinuses, and the paracortex was both small and thinly populated (Fig. 5H). The sharp decline in AZ in the blood two weeks after MPA treatment (Mihok and Schwartz, 1991) was associated with a return to normal lymphocyte numbers in lymphoid organs. Despite an intensive search, we were unsuccessful in discerning the final tissue sink of the AZ. In particular, we found no evidence of uptake of AZ by macrophages. Similarly, we never observed free AZ inclusions in tissue sections, nor did we observe the appearance of cytoplasmic AF + , or PAS + material in cells in any part of the body. Immunomodulation Treatment of voles with either cyclosporin A or hydroxyurea shortly after the injection of MPA resulted in a substantial reduction in AZ. Counts on day 7 were reduced to 39% of control levels with hydroxyurea, and 41% of control levels with cyclosporin A (Fig. 6). Treatment of voles with PGE, or conditioned medium had no effect on AZ. AZ were not induced in any of the treatments that did not receive MPA. As in other experiments, lymphocyte counts were reduced significantly on day 7 when MPA was administered alone (Fig. 7). Administration of cyclosporin A, with or without MPA, resulted in a similar, but non-significant, reduction in lymphocytes. In contrast, administration of hydroxyurea, PGE*, or conditioned medium, resulted in an increase in lymphocytes. These increases were significant in all three treatments involving prior injection of MPA (Fig. 7). Spleen weights on day 7 differed significantly among the treatments (P < O.OOl), but did not differ as a factor of administration of MPA (P > 0.62, 2-way repeated measures ANOVA). Spleens were smallest in voles treated with cyclosporin A, and largest in voles treated with conditioned medium (Fig. 8). Numerous other effects were also noted in voles treated with conditioned medium. On day 3, voles become temporarily anemic (mean drop in

STEVEMIHOK

224

and

hematocrit of about 16 vol %). Anemia was associated with rouleaux formation on blood smears (hypergamma-globulinemia?). Large numbers of platelets, and modest numbers of normoblasts were also characteristic of blood smears on day 3. At autopsy on day 7, numerous histological changes were evident. In addition to a doubling of spleen size (Fig. 8), spleens were densely populated with lymphocytes. Unusually high numbers of plasma cells, megakaryocytes, normoblasts and basophils, were also observed. In the bone marrow, there was an obvious increase in myelopoiesis and megakaryopoiesis. Basophil production was particularly enhanced, averaging 7% of total bone marrow cells in voles treated with conditioned medium (with or without MPA, N = 11) compared with 0.5% in controls (N = 16, from all experiments). Finally, lungs were infiltrated with leukocytes, particularly lymphocytes, monocytes, and basophils. DISCUSSION

Origins and maturation

of the AZ

Our histological studies of mitotic figures in tissues from MPA-treated voles indicate that AZ originate from small-to-medium-sized lymphocytes in the bone marrow. Differentiation of these precursors into AZ was affected by mitotic inhibitors such as cyclophosphamide, hydroxyurea and cyclosporin A, but was insensitive to other biological response modifiers such as estradiol, hydrocortisone, PGE, and conditioned medium (presumably a source of interleukin-2 and other growth factors). Prior to activation, AZ precursors appeared to be quiescent, as prior treatment with cyclophosphamide had no effect. The terminal stages of AZ differentiation probably coincide with the development of small azurophilic granules on about day 3 (Fig. 5b). As AZ in the blood were never observed in mitosis, the bloodstream or “mature” AZ may be the natural endpoint of this cell lineage. Unlike most actively-dividing cells, mature

BILL SCHWARTZ

AZ were insensitive to hydrocortisone or cyclophosphamide (Fig. 2). With our simple histological methods, we were unable to trace the exit sequence of AZ from the bone marrow from about day 3 onwards. AZ appeared simultaneously in the blood, the red pulp of the spleen, and the cortex of the thymus. Based on experiments with dextran sulphate, AZ and their granulated precursors never entered the recirculating lymphocyte population. Although AZ were common in the spleen, their numbers in the blood were neither elevated, nor decreased by splenectomy (Mihok and Schwartz, 1991). We did not attempt thymectomy, and therefore have no information on a possible developmental stage in the thymus. A conventional T-lymphocyte lineage for the AZ seems unlikely, given the insensitivity of AZ induction to hydrocortisone and estradiol (Fig. 1), two agents that have dramatic effects on T-lymphocytes (Fauci, 1978; Luster et al., 1984). Degeneration of lymphoid architecture in the thymus early in AZ induction also makes it an unlikely site for AZ maturation (Fig. SG). Although there are still gaps in our knowledge of how the AZ differentiates, matures, and moves about in the body, we believe that the salient features of its development are now well-defined. Hence, we have presented a qualitative outline of our understanding of cell dynamics in Fig. 9. A quantitative appraisal awaits the application of more sophisticated techniques. Relationships

of AZ to other leukocytes

In our first report on the basic characteristics of the AZ (Mihok et al., 1987), we reviewed the affinities of AZ to other leukocytes and concluded that it might be a NK cell unique to the vole, with special functions related to pregnancy. In humans, and in other rodents, NK activity is thought to be a characteristic of a unique lineage of cells, rather than a function displayed by a diverse lineage (Lanier et al., 1986). NK cells originate in the bone marrow from

Fig. 5. (A) Bone marrow smear of meadow vole 4 hr after treatment with colchicine on day 2 following injection with MPA. Smear contains numerous lymphocytes (ly) in mitosis, as well as small numbers of blast cells (bl) of possible AZ lineage (Azure B-Eosin Y, x 1000). (B) Bone marrow smear of a meadow vole depicted in (A) showing numerous AF + granules in lymphocytes of probable AZ lineage (az), one of which is in mitosis (mi). A blast cell with fine AF + granules (bl) has just completed mitosis. (AF with Neutral Red counterstain, x 1000). (C) Bone marrow smear of‘a meadow vole showing typical staining reactions of myeloid cells. Neutrophils (ne) contain abundant PAS+ cytoplasmic material (glycogen). Earlier cells of the myeloid lineage (my) contain fine AF + granules (primary granules) as well as coarse PAS + material (glycogen). A single basophilic metamyelocyte (ba) contains large AF + specific granules (AB-PAS, phase contrast, x 1000). (D) Bone marrow smear of a meadow vole 7 days after treatment withy cyclophosphamide showing early cells of the myeloid lineage (my). The smear contained mostly promyelocytes and myeloblasts (13%) with a M : E ratio (myeloid: erythroid) of 9: 1 (Azure B-Eosin Y, x 1000). (E) Tissue section from the thymic cortex of a meadow vole treated 9 days earlier with progesterone. AZ with large AF+ inclusions (az) are present next to the septum (AF, phase contrast, x 1000). (F) Tissue section from the spleen of a meadow vole treated 4 days earlier with MPA. Note depletion of lymphocytes from the red-pulp (rp). The white pulp (wp) is also depleted, but still contains active follicles. There is a slight expansion of the marginal zones (mz) (H&E, x 60). (G) Tissue section from the thymus of a meadow vole-4 hr after treatment-with colchicine’on day 3 follow& treatment with MPA. Note the lack of a distinct organization of cells into a cortex and medulla. The section contains numerous pyknotic lymphocytes (small, dark cells), some of which are being engulfed by clusters of macrophages (ma) (H&E, x 250). (H) Tissues section from a mesenteric lymph nodes of a meadow vole treated 7 days earlier with MPA. Note the paucity of cells in the medullary sinuses, and a general depletion of cells from the medullary chords (mc), and the paracortex (pc). The cortex contains numerous germinal centres (gc) with weakly-defined lymphoid cuffs (H&E, x 25).

Azurocyte maturation in the vole

225

Fig. 5

precursors (Kalland, 1986; Migliorati et al., 1987a), and can mature in the absence of an intact spleen or thymus (Ortaldo and Herberman, 1984). The mature cells localize in the blood and spleen, with low levels of activity in the lungs, bone marrow, thymus and lymph nodes (Luini et al., 1981; Itoh lymphoid

et al., 1982). Radiolabelling

studies have confirmed this pattern, and have also shown that the cells do not belong to the recirculating pool of lymphocytes (Reynolds et al., 1984; Rolstad et al., 1986). Altogether, these features of NK cells in other mammals are nearly identical to our findings with AZ.

226

STEVEMIHOKand BILLSCHWARTZ Azurocytes

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z o

1

0.0

Fig. 6. Absolute changes in AZ counts on day 7 in meadow voles treated with various drugs after treatment with MPA. Asterisks denote values significantly different from controls receiving MPA only.

Although many agents can induce NK cells (Ortaldo and Herberman, 1984), researchers have concentrated primarily on the role of interferon and interferon inducers (Huntley et al., 1984; Damle et al., 1986; Santoni et al., 1985; Testi et al., 1986; Timonen and Pakkanen, 1987; Kaminsky et al., 1987; Migliorati et al., 1987b; Maghazachi et al., 1988; McIntyre et al., 1988; Powell et al., 1989). These studies have revealed a variety of developmental pathways and sites of action for NK cells; a discussion of which is beyond the scope of this paper. Nevetheless, when these results are compared with our studies of the AZ, it is obvious that the AZ differs from NK cells in a few important ways. In particular, the induction of AZ through progestins appears to have no counterpart in the immunobiology of NK cells. Similarly, interferon inducers, which have rapid and potent effects on NK cell induction in other mammals, have slow and weakly-developed effects on AZ induction in voles (Mihok and Schwartz, 1991). Finally, we are puzzled by our inability to stimulate AZ maturation with a crude preparation of interleukin-2, an agent that is quite effective in inducing NK activity in other mammals. The dependence of AZ induction on a steroid hormone is unusual; the only similar process appears to be the estrogenic induction of KC in guinea-pigs. Lymphocytes have binding sites for corticosteroids, but lack similar sites for estrogens, androgens and progestins. The effects of these hormones are thereLymphocytes

3

on Day 7

fore thought to be mediated through interactions with a specific glucocorticoid receptor (Stimson et al., 1983). Although past attempts to find an estrogen receptor on KC have failed (Landemore et al., 1983), Landemore et al. (1988) have recently found lowaffinity estrogen binding sites. Other workers have, however, failed to culture KC from various tissues by incorporating estradiol or serum from estradioltreated animals in media (Sandberg and Hagelin, 1986) We were also unsuccessful in a preliminary attempt to induce AZ with tissue-transfer experiments (Mihok and Schwartz, 1991). Recently, Thomson et al. (1988) have achieved a modest breakthough in the immunobiology of the KC that may be of significance for the general family of NK cells. They succeeded in inducing remarkable levels of KC in guinea-pigs without estrogenic stimulation through sequential treatment with cyclophosphamide, an immunostimulant (ovalbumin in complete Freunds Adjuvant), and cyclosporin A. A similar protocol also effectively induced the classic NK cells (LGL) found in rats (Mathie et al., 1987). These results suggest that B-lymphocytes (cyclophosphamide-sensitive) and T-lymphocytes (cyclosporin A-sensitive) may act together to suppress NK cell maturation. Estrogenic stimulation of KC may therefore be an indirect process, involving the release of suppressive effects of regulatory cells, such as T-suppressor lymphocytes (Stimson and Hunter, 1980; Stimson, 1988). We find this hypothesis intuitively attractive for AZ, given the lymphoid origin of the AZ in the bone marrow, its localization in T-lymphocyte areas of the body, and its sensitivity to induction by non-specific interferon inducers. We are also intrigued by an earlier report showing the induction of “atypical” lymphocytes resembling AZ or LGL after 21 days of cyclosporin A treatment in rats (Thomson et al., 1981). Thomson’s protocols are clearly worth testing in voles, despite our results concerning cyclosporin A-induced depression of AZ induction. Function of the AZ

The main puzzle remaining in the immunobiology of the AZ is its function. From the association of AZ with pregnancy in the wild (Mihok et al., 1987), we would assume a priori that the cell is required during pregnancy for maintaining immunocompetence, or is perhaps involved in maintaining survival of the fetal allograft. Why the AZ is a feature of pregnancy in

3

Conditionad

2

Spleens

2 100

WI

5 0

1

1

0

0

-1

-1

-2

-2 m

MPA + Drug

m

I

on Day 7

ConditIoned Madlum

‘;;;

60

60

5

60

60

i .P

40

40

5

20

20

Drug Alono

0 Fig. 7. Absolute changes in lymphocyte counts on day 7 in meadow voles treated with various drugs after treatment with MPA. Asterisks denote values significantly different from controls receiving MPA only.

0 D

MPA+ Drug mTI Drug Alone

Fig. 8. Spleen size on day 7 in meadow voles treated with various drugs after treatment with MPA.

Azurocyte maturation in the vole

BM

Cyclosporin Hydroxyurea Inhibition

227

A

I

THYMUS

1

T1 IBLooDI I

I

I

I

Days

of

Induction

Day

+

Hydrocortisone/Estradiol

7+

Insensitive

Fig. 9. Schematic representation of the effectsof various biological response modifiers on AZ development in the meadow vole (Microtuspenmyhanicus). Microtus, and not in closely-related genera, is a mystery. Pregnancy in Microtus is qualitatively similar to pregnancy in other cricetids (Seabloom, 1985). Similarly, large changes in NK cell levels are not a normal feature of mammalian pregnancy (Baley et al., 1985; Gregory et al., 1987). Pregnancy in mammals is invariably associated with increased levels of progesterone; at very high concentrations this hormone has clear immunosuppressive and anti-inflammatory actions (Siiteri et al., 1977; Siiteri and Stites, 1982). Although experimentally-proven, these effects have been difficult to demonstrate at physiological levels. Hence, there is little evidence for a decreased immune responsiveness in pregnancy associated with acceptance of the fetal allograft (Gill, 1985). If anything, the uterus appears to be a site of intense immunological activity during pregnancy, with increases in many immune cells (Hunt et al., 1985; Kearns and Lala, 1985; Parr and Parr, 1985). How the fetal allograft protects itself from this activity is poorly-understood (Hunziker and Wegmann, 1986). Ideally, insight into the function of AZ should be based on studies of pregnant voles. This is, however, difficult to accomplish with most species of Microtus, as they do not breed well in captivity. Our inability to produce high numbers of AZ with the natural hormone progesterone (Mihok et al., 1987; Mihok and Schwartz, 1991) prompted us to concentrate on a synthetic analogue, MPA. Some side-effects of this drug non-specific to AZ induction may therefore have been incorporated in our work. In particular, inconsistent patterns in lymphocyte numbers with various drugs (Mihok et al., 1991), suggest that the severe lymphopaenia observed with MPA treatment may not be associated reliably with AZ function. While AZ were present in the peripheral blood, lymphocytes and other leukocytes responded normally to most biological response modifiers. For example, cytotoxic agents such as hydrocortisone and cyclophosphamide caused a rapid decrease in lymphocytes, irrespective of the time that these drugs were administered (Fig. 4). Similarly, conditioned

medium had a prounounced stimulatory effect on many cell lines, including lymphocytes (Fig. 7). Overall, we noted only two puzzling trends: hydroxyurea and PGE, caused unexpected increases in lymphocytes when administered in combination with MPA. Nevertheless, these effects may have been unrelated to AZ induction, as the two drugs had different effects on the number of AZ induced by MPA (Fig. 6). In humans and other rodents, high dose of MPA usually have some effect on cellular or humoral immune responses (Spreafico et al., 1982; Bell et al., 1983; Gronroos and Eskola, 1984). However, these effects do not appear to be as dramatic as those observed in the vole. From our histological studies, one probable site of action for the AZ outside of the blood appears to be the thymus. The thymus reacted dramatically to MPA treatment (Fig. 5G), and was the only organ where AZ localized and accumulated in a specific pattern. The cells were found in small clusters along septa, and were usually associated with the outer cortex, or the cortico-medullary junction. Hence, they were situated in an ideal position to affect the early stages of T-lymphocyte maturation. KC in guineapigs also localize in the thymus, and may complete the final stages of their development in this organ (Sandberg and Hagelin, 1986). The thymus is an intriguing site for the localization of cells that are induced by steroid hormones. In mice, T-lymphocytes possess an enzyme (20a-hydroxysteroid dehydrogenase) that converts progesterone to 20a-dihydroprogesterone. The lineage that possesses this enyzme in regulated by interleukin-3. Under its influence, this lineage matures into a granulated mast-like cell that possesses natural cytotoxicity against adherent cell targets, but not lymphoid targets (Ihle and Weinstein, 1985). For unknown reasons, the enzyme, and presumably the lineage associated with it, is absent in other mammals. Although we do not yet know the exact affinities of AZ to KC, LGL, lymphokine-activated killer cells, cytotoxic lymphocytes, and similar cells, we feel

228

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confident that the AZ falls within this general family of cytotoxic cells. The AZ is obviously unique in many ways, and hence, may simply be something new. Now that many features of the cell are known, we suggest that studies of cellular cytotoxicity, 20a-hydroxysteroid dehydrogenase activity, and T-suppressor lymphocyte activity would best help elucidate the function of the cell. Hopefully, our

their relationship to mucosal mast cells and globule leucocytes in the rat. Immunology 53, 525-535. Hunziker R. D. and Weamann T. G. (1986) Placental immunoregulation. CRC-Crit. Rev. Imminol. ii, 245-285. ICSH (1984) ICSH reference method for staining blood and bone marrow films by Azure B and Eosin Y (Romanowsky stain): International Commission for Standardization in Haematology. Br. J. Haematol. 57,

summary of the cell’s basic characteristics will stimulate further interest in the immunobiology of this enigmatic cell.

Ihle J. N. and Weinstein Y. (1985) Interleukin 3. Regulation of a lineage of lymphocytes characterized by the expression of 20a SDH. In Recognition and Regulation in Cell-Mediated Immunity (Edited by Watson J. D. and Marbrook J.), pp. 291-324. Marcel Dekker, New York. Itoh K., Suzuki R., Umezu Y., Hanaumi K. and Kumagai K. (1982) Studies of murine large granular lymphocytes II. Tissue, strain and age distributions of LGL and LAL.

Acknowledgements-This

work was conducted while the senior author was a research scientist with Atomic Energy of Canada Limited. AECL’s funding of basic research is gratefully acknowledged. The authors wish to thank Todd Lawton, Val Kidson. Marlene Goodwin and Malcolm Sargent to technical assistance. REFERENCES Baley J. E. and Schacter B. Z. (1985) Mechanisms of diminished natural killer cell activity in pregnant women and neonates. J. Immunol. 134, 3042-3055.Bell F. E.. Palmer J. S. and Dawson W. D. (1983) Medroxvprogesterone, immunosuppression and conceptus size h Peromyscus. J. exp. 2001. 226, 273-219.

Bradfield J. W. B. and Bor G. V. R. (1974) Lymphocytosis produced by heparin and other sulphated polysaccharides in mice and rats. Cell. Immunol. 14, 22-32. Buat M.-L., Landemore G. and Izard J. (1988) Alphanapthyl acetate esterase activities in guinea-pig Kurloff cells, a cytochemical and electrophoretic study. J. Histochem. Cytochem. 36, 1109-l 115. Bulmer D., Peel S. and Stewart I. (1987) The metrial gland. Cell D~fl. 20, 77786. Chang A. E., Hyatt C. L. and Rosenberg S. A. (1984) Systemic administration of recombinant human interleukin-2 in mice. J. biol. Resp. Mod. 3, 561-572. Damle N. K., Doyle L. V. and Bradley E. C. (1986) Interleukin 2-activated human killer cells are derived from phenotypically heterogeneous precursors. J. Immunology 137, 2814-2822.

Fauci A. S. (1978) Mechanisms of the immunosuppressive and anti-inflammatory effects of glucocorticosteroids. J. Immunopharmacol. 1, l-25. Gentile P., Byer D. and Pelus L. M. (1983) In vivo modulation of murine myelopoiesis following intravenous administration of prostaglandin E,. Blood 62, 1100-l 107. Gill T. J. III. (1985) Immunity and pregnancy. CRC Crit. Rev. Immunol. 5, 201-228.

Gregory C. D., Lee H., Scott I. V. and Golding P. R. (1987) Phenotypic heterogeneity and recycling capacity of natural killer cells in normal human pregnancy. J. Reprod. Immunol. 11, 135-145. Griinroos M. and Eskola J. (1984) In vitro functions of lymphocytes during high-dose medroxyprogesterone acetate (MPA) treatment. Cancer Immunol. Immunother. 17, 218-220.

Herberman R. B., Reynolds C. W. and Ortaldo J. R. (1986) Mechanism of cytotoxicity by natural killer (NK) cells. A. Rev. Immunol. 4, 651d80.

Hodgson G. S., Bradley T. R., Martin R. F., Sumner M. and Fry P. (1975) Recovery of proliferating haemopoietic progenitor cells after killing by hydroxyurea. Cell Tissue Kinet. 8, 5160. Hunt J. S., Manning L. S., Mitchell D., Selanders J. R. and Wood G. W. (1985) Localization and characterization of macrophages in murine uterus. J. Leuk. Biol. 38, 255-265.

Huntley J. F., McGorum B., Newlands G. F. J. and Miller H. R. P. (1984) Granulated intraepithelial lymphocytes,

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Kalland T. (1986) Generation of natural killer cells from bone marrow ’ precursors in vitro. Immunology 57, 493498.

Kaminsky S. G., Milisauskas V., Chen P. B. and Nakamura I. (1987) Defective differentiation in natural killer cells in SJL mice. Role of the thymus. J. Immunology 138, 1020-1025. Kearns M. and Lala P. K. (1985) Characterization of hematogenous cellular constituents of the murine decidua, a surface marker study. J. Reprod. Immunol. 8, 213-224.

Landemore G., Buat M.-L. and Izard J. Z. (1987) Zymograms of Kurloff cell acid phosphatases, thin layer isoelectric focusing and native polyacrylamide 415% gradient gel electrophoresis. Biol. Cell. 59, 97-100. Landemore G., Darbon J.-M. and Izard J. (1983) Lack of estradiol receptor in Kurloff cell cytosol. Mol. Cell. Endocr. 30, 353-355.

Landemore G., Darbon J.-M., Izard J. Bayard F. and Faye J.-C. (1988) Presence of low-affinity estrogen binding sites in guinea-pig Kurloff cells. J. Steroid Biochem. 31, 5740.

Lanier L. L., Phillips J. H., Hackett J. Jr., Tutt M. and Kumar V. (1986) Natural killer cells: definition of a cell type rather than a function. J. Immunol. 137, 2735-2739.

Luini W., Boraschi D., Alberti S., Aleotti A. and Tagliabue A. (1981) Morphological characterization of a cell population responsible for natural killer activity. Immunology 43, 663668.

Luster M. I., Hayes H. T., Korach K., Tucker A. N., Dean J. H., Greenlee W. F. and Boorman G. A. (1984) Estrogen immunosuppression is regulated through estrogenic responses in the thymus. J. Immunol. 133, 110-116.

Maghazachi A. A., Vujanovic N. L., Herberman R. B. and Hiserodt J. C. (1988) Lymphokine-activated killer cells in rats-IV. Developmental relationships among large agranular lymphocytes, large granular lymphocytes and lymphokine-activated killer cells. J. Immunol. 140, 28462852. Mathie I. H., Sewell H. F. and Thomson A. W. (1987) Generation of large granular lymphocytes and lymphocyte subset changes linked with cyclophosphamideinduced eosinophilia in rats, and the effects of ciclosporin. Stand. J. Immunol. 26, 417-423.

Mayrhofer G. (1980) Fixation and staining of granules in mucosal mast cells and intraepithelial lymphocytes in the rat jejunum, with special reference to the relationship between the acid glycosaminoglycans in the two cell types. Histochem. J. 12, 513-526.

McIntyre K. W., Natuk R. J., Biron C. A., Kase K., Greenberger J. and Welsh R. M. (1988) Blastogenesis of large granular lymphocytes in nonlymphoid organs. J. Leuk. Biol. 43, 492-50 1.

Azurocyte maturation in the vole Migliorati G., Cannarile L., D’Adamio L., Herberman R. B. and Riccardi C. (1987a) Interleukin-1 augments the Interleukin-Zdependent generation of natural killer cells from bone marrow precursors. Nat. Immun. Cell Growth Regul. 6, 306-315.

Migliorati G., Cannarile L., Herberman R. B., Bartocci A., Stanley E. R. and Riccardi C. J. (1987b) Role of interleukin 2 (IL 2) and hemopoietin-1 (H-l) in the generation of mouse natural killer (NK) cells from primitive bone marrow precursors. Immunology 138, 3618-3625. Mihok S. (1987) Pregnancy test for meadow voles (Microtus pennsylvanicus) based on blood azurocyte counts. Can. J. Zool. 65, 283&2832. Mihok S., DescGteaux J.-P., Lawton T., Lobreau A. and Schwartz B. (1987) The azurocyte: a new kind of leukocyte from wild volks (Microtusj. Can. J. Zool. 65, 54-62. Mihok S. and Schwartz B. (1991) Artificial induction of azurocytes in the meadow vole (Microtuspennsylvanicus). Comp. Biochem. Physiol. WC, 213-218.

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Santoni A., Piccoli M., Ortaldo J. R., Mason L., Wiltrout R. H. and Herberman R. B. (1985) Changes in number and density of large granular lymphocytes upon in uivo augmentation of mouse natural killer activity. J. Immunology 134, 2799-2810. Sarneva M., Vujanovic N. L., Van den Brink M. R. M., Herberman R. B. and Hiserodt J. C. (1989) Lymphokineactivated killer cells in rats: generation of natural killer cells and lymphokine-activated killer cells from bone marrow progenitor cells. Cell. Immunol. 118, 448-457. Seabloom R. S. (1985) Endocrinology. In Biology of New World Microtus (Edited by Tamarin R. H.), pp. 685-724. Special Publication No. 8, American Society of Mammalogists, Pittsburgh. Siiteri P. K., Febres F., Clemens L. E., Chang R. J., Gondos B. and Stites D. P. (1977) Progesterone and maintenance of pregnancy: is progesterone nature’s immunosuppressant? Ann. N. Y. Acad. Sci. 286, 384-397. Siiteri P. K. and Stites D. P. (1982) Immunologic and endocrine interrelationships in pregnancy. Biol. Reprod. 26, I-14.

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APPENDIX Staining AZ in para&-embedded

sections

With H&E staining, AZ inclusions are weakly eosinophilic and are not readily visible at low magnification. Identification is therefore best done by staining for acidic glycosaminoglycans with stains such as AF or AB. AF is recommended for critical work as it stains the inclusions slightly better than AB. PAS can be used to stain the glycoproteins present in the inclusions, but it is not recommended alone, as it stains a wide variety of cellular materials. However, a combined AB-PAS stain is adequate for an overview of cell types. AF can also be combined with a weak counterstain of Neutral Red, Light Green, or Acid Fuchsin. AF or AB staining of glycosaminoglycans requires alcoholic fixatives, such as MFAA, AF, AB and PAS _._staining are also satisfactory in material fixed with Carnoy and Mota fixatives. Adding 1% cetylpyridinium chloride to MFAA does not improve staining. Differentiating AZ from similar ceils

AZ inclusions stain deep purple with AF, blue with AB, magenta with PAS, and blue with AB-PAS. Other cells with similar morphology or staining properties can be identified based on the following characteristics. (1) Granulated intraepithelial lymphocytes: these are found in the small intestine and are similar in morphology to AZ. However, they have fewer inclusions that stain weakly with AF/AB, and stain magenta with AB-PAS. (2) Macrophages: These are ubiquitous in lymphoid tissues; they can be identified through their large size, complex nuclear morphology, or the presence of phagocytosed material. Their inclusions are larger than those in AZ, and are AF/AB -, PAS+, and stain magenta with AB-PAS. Macrophages from the spleen, lymph nodes,

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STEVEMIHOKand BILLSCHWARTZ

and peritoneal cavity sometimes contain fine azurophilic granules that are AF/AB + (3) Metrial gland cells: these cells are about twice as large as AZ. They are found in the uterus during pregnancy only. With MFAA fixation, they do not stain with AF/AB, but are PAS+, and hence stain magenta with AB-PAS. With other fixatives, AF/AB staining varies from weak to intense; PAS staining is negative or is weak. AB-PAS staining can result in magenta, blue, or purple colors depending on the fixative. (4) Mast cells: these are ubiquitous in connective tissue, and are present in small numbers in the spleen and lymph nodes. They can usually be distinguished on morphological grounds unless fixation is poor. They contain abundant, fine AF/AB+, PASgranules that stain blue with A&PAS. AB staining is maintained at high Mg concentrations. (5) Basophils: these are present in lymphoid tissues in small numbers; they are easily identified through

morphology in smears, but are difficult to identify in tissue sections. In well-prepared material, basophils can be identified on the basis of granule shape (elliptical), and nuclear morphology. Basophil granules and AZ inclusions are PAS + and AF/AB+ In air-dried smears, the cells can be differentiated with the a-napthyl-AS-D chloroacetate esterase reaction. Basophil granules are strongly positive, whereas AZ inclusions are mostly negative, or only weakly positive. (6) Reticular cells?: the medullary sinuses of vole lymph nodes contain numerous large cells with weakly eosinophilic cytoplasm. The cells are 2-3 x the size of AZ and contain indistinct inclusions that give the cytoplasm a “foamy” appearance. The cytoplasm stains routinely with PAS, sometimes intensely. In contrast, staining with AF/AB varies from negative to intensely positive. Hence, the cells stain magenta, blue or purple with AB-PAS. These cells contain the most brilliant purple AB-PAS reaction observed in any cell in the body.