Immunology 1985 54 589
The heterogeneity of mononuclear phagocytes in lymphoid organs:
distinct macrophage subpopulations in the rat recognized by monoclonal
antibodies ED1, ED2 and ED3
C. D. DIJKSTRA,* E. A. DOPP,* P. JOLINGt & G. KRAAL* *Department ofHistology, Free University,
Amsterdam and tDepartment of Internal Medicine I, Erasmus University, Rotterdam, The Netherlands
Acceptedfor publication 16 October 1984
Summary. In the present study, a set of three monoclonal antibodies is described, each of which recognizes cells of the monocyte-macrophage lineage in the
rat. The tissue distribution, in particular in lymphoid
organs, of each of the three monoclonals is determined
by immunoenzyme histochemistry on cryostat sections, as well as on cell suspensions. Results show that
ED1 recognizes a cytoplasmic antigen in monocytes
and in most macrophages, free and fixed. ED2 and
ED3 recognize membrane antigens of tissue macrophages, discriminating between distinct subpopulations of macrophages, each with a characteristic
localization in the compartments of lymphoid organs.
No other cell types except cells of the mononuclear
phagocyte system are positive for any of the three
monoclonals. Possible relations between the macrophages recognized by this set of monoclonals and
dendritic cells are discussed.
INTRODUCTION
The precursors of macrophages originating in the
Abbreviations: FDC, follicular dendritic cell; IDC, interdigitating cell; i.p., intrapentoneally; PALS, periarteriolar
lymphocyte sheath.
Correspondence: Dr C. D. Dijkstra, Department of Histology, Medical Faculty, Free University, P.O. Box 7161,
1007 MC Amsterdam, The Netherlands.
bone marrow differentiate into circulating monocytes
which, after leaving the peripheral blood for the tissue
compartments, differentiate into macrophages (Van
Furth et al., 1972). The macrophages form a heterogeneous population, as expressed by differences in
their characteristics, such as morphology, density,
enzyme activity, cell surface properties and functional
capacities. Together, these characteristics determine
the phenotypic appearance of macrophages, which
differs with different environmental circumstances,
such as the state of activity (Cohn, 1978; Morahan,
1980; Walker, 1976; Beelen, Broekhuis-Fluitsma &
Hoefsmit, 1978), the developmental stage of the
individual macrophages (Cohn & Benson, 1964), the
localization in different tissues (Twomey, Laughter &
Brown, 1983; Flotte, Springer & Thorbecke, 1982;
Haines et al., 1983) and compartments (Hoefsmit et
al., 1980) and often a combination of these factors.
Whether these environmental conditions determine
the phenotypic appearance of the macrophages (Sorg,
1982) or the origin of the heterogeneity is already
determined at bone marrow level (Bursuker & Goldman, 1983) is still a subject of discussion.
MATERIALS AND METHODS
Animals
Wistar rats and BALB/c mice were obtained for
589C. D. Dijkstra et al.
Centraal Proefdierenbedrijf (T.N.O., Zeist, The Netherlands and kept under routine laboratory conditions.
Preparation of the monoclonal antibodies
BALB/c mice were primed intraperitoneally with rat
spleen cells and Freund's complete adjuvant and
boosted i.p. after 4 weeks with rat spleen cells and
Freund's incomplete adjuvant. Four days after the last
immunization, two mice were killed, their spleen cells
were pooled and fused with Sp2/0-agl4 myeloma cells
according to the method of Kohler & Milstein (1975),
modified after Fazekas de St Groth & Scheidegger
(1980). The fusion mixture was plated in 96-well
culture trays (Costar) with BALB/c peritoneal exudate
cells as feeder cells. Hybrids surviving in hypoxanthin,
aminopterin and thymidin (HAT) selective
RPMI- 1640 medium, supplemented with 15% fetal
calf serum (FCS, (Hy Clone), Greiner, Alphen, The
Netherlands) were tested. Screening of the monoclonal antibodies was performed on cryostat sections of rat
spleen by a two-step immunoperoxidase method using
peroxidase conjugated rabbit anti-mouse Ig (Dako,
Denmark) as second antibody (Dijkstra, 1982). The
procedure is described under 'Immuno- and enzymehistochemistry'. Clones which reacted with putative
macrophages in the spleen sections were selected and
cloned. Ascites fluid was obtained by intraperitoneal
injection of 3 x 106 hybrid cells into BALB/c mice.
Immuno- and enzyme-histochemistry
The following organs were frozen in liquid nitrogen
and stored at - 200: spleen, thymus, mesenterial
lymph node, Peyer's patches, lung (including BALT),
liver and bone marrow. Cryostat sections of 8-12 pm
were picked up on slides and air-dried. Sections were
fixed in acetone for 10 min and air-dried for at least 30
min. Sections were then incubated for 60 min with
culture supernatant or with ascites fluid, diluted
1: 3000. After washing in 0-01 M phosphate-buffered
saline (PBS, pH 7.4), slides were covered with a 1: 200
dilution of rabbit anti-mouse Ig peroxidase (Dako) in
PBS with 0 2% BSA and 1% normal rat serum for 60
min. After washing in PBS, the slides were stained for
peroxidase activity with 3,3'-diaminobenzidine-tetrahydrochloride (DAB, Sigma, St Louis, MO) (0-5
mg/ml Tris-HCl buffer, pH 7 6, containing 0-01%
H202). Control slides were incubated in the same way
omitting the first step. Where desired, endogenous
peroxidase activity was eliminated by incubation in
methanol with 0 03% H202 (Streefkerk & van der
Ploeg, 1974).
Acid phosphatase activity was demonstrated
according to Burnstone (Pearse, 1968) with naphthol
AS-BI phosphate (Sigma) as substrate and hexazotized pararosaniline as diazonium salt. This procedure
was carried out on the cryostat sections following the
immunoperoxidase procedure.
Plastic embedded material
Perfusion fixation through the aorta with 0-2% glutaraldehyde and 1% N(3-di-methylaminopropyl)-N'-ethylcarbodiimidhydrochlorid (Merck, Darmstadt,
FRG) was performed. Lymphoid organs were
removed, cut in pieces of about 0 5 cm thickness, and
fixed in the same solution for another 2-3 h at 4°.
Vibratome sections of 100 pm thickness were then
prepared, washed and incubated subsequently in the
antibody (24 hr), conjugate (24 hr) and DAB (1 hr
without H202, 1 hr with H202) solution as described
above for the cryostat sections. After postfixation with
OS04, the 100 pm sections were dehydrated and
embedded in araldite. Selected areas were trimmed
out, I pm-thick sections prepared on an LKB Ultramicrotome.
Cell suspensions
Lymphoid organs. Spleen, mesenteric lymph node
and thymus were cut into small pieces, gently pressed
through nylon gauze and suspended in Earl's medium
(NPBI, Emmer-Compascuum, The Netherlands).
Peripheral blood. Two to three ml of blood was
obtained via cardiac puncture and decoagulated with
heparin. Erythrocytes were removed by ammonium
shock.
Bone marrow. The femur was excised, the ends cut
off, and the marrow eluted and suspended in Earl's
medium.
Peritoneal cavity. Peritoneal macrophages were
harvested after intraperitoneal (i.p.) injection of 10 ml
Earl's medium, which was recollected after 5 min.
These cell suspensions were used to prepare cytocentrifuge preparations; in order to perform glassadherence, preparations were stained with the monoclonal antibodies, followed by demonstration of acid
phosphatase activity. Positive cells were counted and
related to the total population of macrophages, as
judged by morphology and acid phosphatase activity.
590Heterogeneity of lymphoid mononuclear phagocytes
Veiled (dendritic) cells
Cell suspensions of spleen and lymph node were
enriched for veiled cells according to Knight et al.
(1983), using a metrizamide (analytical grade, Nyegaard, Norway) sugar gradient. In cytocentrifuge
preparations, cells with characteristics of dendritic
cells (irregular shaped, excentric nucleus, extensive cell
processes, acid phosphatase activity restricted to a
distinct area in the cytocentre) were judged for
positivity for either of the three monoclonals.
Determination of isotype
The isotype of the monclonal antibodies was determined in a two-step immunoperoxidase method on
cryostat sections of rat spleen using peroxidase conjugates specific for each of the mouse immunoglobulins
isotypes (Serotec, Oxford, U.K.) as second step.
RESULTS
EDI, ED2 and ED3 are specific for macrophages
The ED 1, ED2 and ED3 monoclonal antibodies all
reacted with large, round oval cells or with cells with a
dendritic appearance by their slender cell processes.
These cells always showed acid phosphatase activity in
every organ tested, as demonstrated by combination
of the immunoperoxidase procedure with the
demonstration of acid phosphatase activity in the
same cryostat section. In the splenic red pulp, as well
as in the white pulp, cells positive for any of the three
monoclonal antibodies contained latex particles 24 hr
after intravenous injection (e.g. Fig. Id). These findings, together with the localization pattern, indicate
that these monoclonal antibodies recognize macrophages. They did not recognize other cells, such as
granulocytes, lymphocytes, follicular dendritic cells,
vascular endothelium, mucosal or bronchial epithelium or liver parenchymal cells.
The distribution and staining pattern of macrophages recognized by each of the three monoclonals is
summarized in Table 1. The ED1 monoclonal antibody showed on cryostat sections a granular staining
pattern within the cytoplasm of cells. ED2 and ED3
showed a more diffuse staining pattern, not only on the
cell bodies but also on the blunt or dendritic cell
processes (Fig. Id, Fig. 2b). This staining pattern is
suggestive for membrane staining. The heavy chain
class of EDI is IgGI, and that of ED2 and ED3 is
IgG2a. Some relevant findings in different lymphoid
organs are described below.
Staining patterns and localization
A. Spleen (Fig. 1)
The red pulp macrophages are positive for all three
antibodies. ED3, however, stained with red pulp
macrophages weakly, the staining intensity increased
towards the white pulp (Fig. Id). The macrophages in
the marginal zone and at the inner border of the
marginal sinus were strongly positive for ED3, and
negative for ED2. ED2 stained a few cells or cell
processes in the outer periarteriolar lymphocyte
sheath. A patchy staining pattern throughout the
white pulp, except in the follicles, was seen with ED3.
The distribution in the PALS of ED3-positive cells was
similar to that of the Ia-positive interdigitating cells
(Dijkstra, 1982).
B. Lymph node (Fig. 2)
All medullary macrophages of lymph nodes are
positive for EDl and ED3, only a few are positive for
ED2 (Fig. 2). The subsinusoidal macrophages were
strongly positive for ED3 (Fig. 2b) and negative for
ED2. These branched cells formed a continuous rim at
the inner side of the marginal sinus. ED2 stained
spindle-shaped cells in the lymph node capsule.
C. Peyer's patches
Macrophages in the subepithelial tissue of the intestinal villi stained with ED1 and ED2. ED 1 revealed a
patchy staining pattern in the interfollicular area
(IFA) of the Peyer's patches, ED2 stained branched
cells in the IFA and a few cells in the dome area, ED3
stained only small groups of 1-4 cells at the base of the
IFA. At the mesothelial side, a rim of spindle-shaped
cells stained with EDl and ED2.
D. Thymus (Fig. 3)
The most conspicious staining pattern in the thymus
was observed after staining with ED2 (Fig. 3a). In the
cortex, large cells were found with cell processes
extending between the surrounding lymphocytes (Fig.
3b). These cells showed strong acid phosphatase
activity; only very few cells with acid phosphatase
activity in the cortex did not stain with ED2. The
medulla was absolutely negative for ED2. ED 1
showed a patchy staining pattern in cortex and
medulla, the highest concentration of patches was
found in the corticomedullary region (Fig. 3a). ED3
stained only a few cells very weakly in the thymic
medulla.
591C. D. Dijkstra et al.
Figure 1. Cryostat sections of rat spleen stained for either of the three monoclonal antibodies in an immunoperoxidase
procedure. (a) ED 1: a patchy staining pattern is found in the PALS, follicle and marginal zone. The red pulp macrophages show
heavy staining for EDI (magnification x 80). (b) ED2: all red pulp macrophages are stained, as well as a few cells in the outer
PALS (magnification x 80). (c) ED3: a rim of strongly positive cells is present at the periphery of the PALS and follicle.
Furthermore, large cells with blunt cell processes in the marginal zone are stained. Red pulp macrophages are only weakly
positive (magnification x 80). (d) ED3: (in detail). The ED3-positive cells show blunt and dendritic cell processes. The
phagocytized latex particles can be easily recognized, especially in the weakly stained cells (magnification x 320). PALS,
periarteriolar lymphocyte sheath; f, follicle; mz, marginal zone; rp, red pulp.
592Heterogeneity of lymphoid mononuclear phagocytes
Table 1. Distribution and staining pattern of macrophages identified by EDI, ED2 and ED3 in various organs
Monoclonal antibody EDI ED2 ED3
Isotype IgGI IgG2a IgG2a
Staining pattern Granular, patchy Diffuse, membrane Diffuse, membrane
cytoplasmic
Spleen
White pulp
inner PALS + + +Weakly
outer PALS + + + +Weakly
follicle -+
marg. metallophils -/ + Weakly + + + Branched
marginal zone + Weakly + + + Branched
Red pulp + + + + + + + + + Weakly
Lymph node
Cortex
outer cortex + Weakly + + + subsinusoidal branched
paracortical area + + +
follicles -/ +
Medulla + + + +l10200% + + +
Capsule + +
Peyer's patches
Interfollicular area + + + + + + Small groups 3-4 cells
Dome +
Follicle
Villi + + + Apex + + Apex basis
Lung
BALT + + Only at periphery of BALT
Perivascular/peribronchial + + + +
Alveolar + + +
Thymus
Cortex + + + + Branched
Medulla + + -/+ Weakly
Corticomedullary area + + + + + +
Capsule + + + Branched + + + Branched + + Branched
Liver + + + Branched + + + Branched + + Branched
Bone marrow + + + Monocytes/macrophages + + Macrophages
+ + + = (Almost) all acid phosphatase-positive cells stained with the monoclonal antibody.
+ + = A considerable number stained.
+ = Few stained.
-/+ = Very few stained, or none at all.
E. Lung
In the bronchus-associated lymphoid tissue (BALT),
large branched cells were stained by ED1. In the
perivascular and peribronchial tissue, as well as at the
periphery of the BALT, branched cells, strongly
positive for ED2 were found. The alveolar macrophages were strongly positive for ED1 (Fig. 4), and
negative for ED2 and ED3.
F. Liver
EDI and ED2 recognize branched cells in the sinuses,
probably the cells of Von Kupffer. A smaller number
of these cells was recognized by ED3.
G. Bone marrow
ED1 stains a considerable number of cells scattered
through the bone marrow, comprising large macrophages as well as small monocyte-like cells. ED2
reveals large cells in the bone marrow, often surrounded by erythropoietic cells, together forming a
sort of island. ED3 was negative on cryostat sections
of bone marrow.
ED1 recognizes a cytoplasmic antigen, ED2 and ED3
recognize a membrane antigen
In plastic sections of lymph node medulla, it was clear
593C. D. Dijkstra et al.
I;
FIgure 2. Lymph node. (a) ED1: a patchy staining pattern is found in the cortex; the medullary macrophages show heavy staining
for EDI (magnification x 80). (b) ED2: only very few cells in the lymph node medulla are positive for ED2. Furthermore, some
spindle-shaped positive cells are present in the fibrous capsule (magnification x 80). (c) ED3: all medullary macrophages are
strongly positive for ED3. Furthermore, a rim ofdendritic cells is present at the inner border ofthe marginal sinus (magnification
x 80). (d) ED3: detail of the corticomedullary border, dendritic positive cells are visible at the inner border ofthe marginal sinus
and in the medulla of the lymph node (magnification x 320). C, cortex; M, medulla.
that the antigen recognized by EDI was located within
the cytoplasm ofmacrophages, whereas ED2 and ED3
showed a membrane localization (Fig. 5).
Cell suspensions
The results on cell suspensions are summarized in
Table 2. In bone marrow, monocyte-like cells as well
as macrophages were positive for ED 1, whereas ED2
stained only macrophages. Monocytes in peripheral
blood were positive for EDI and negative for ED2 and
ED3. Peritoneal resident macrophages were strongly
positive for ED1, 45% stained only weakly with ED2.
In cell suspensions enriched for veiled cells (Knight et
al., 1983), almost all cells with characteristics of
dendritic cells were stained by EDI, mostly in a
distinct area in the cytocentre.
DISCUSSION
In this study, three monoclonal antibodies have been
described which recognize different antigenic determinants of rat tissue macrophages. The fact that these
594Heterogeneity of lymphoid mononuclear phagocytes
Figure 3. Thymus. (a) ED1: a patchy staining pattern is found in all thymic compartments, the corticomedullary border can be
recognized by a relative crowding ofpositive cells (magnification x 80). (b) ED2: positive dendritic cells are present in the thymic
cortex, the medulla does not contain ED2-positive cells (magnification x 80). (c) ED2 (in detail): the ED2-positive cells in the
thymic cortex show extensive dendritic cell processes in intimate contact with the surrounding lymphocytes (magnification
x 320). C, cortex; M, medulla.
595C. D. Dijkstra et al.
Figure 4. Lung. ED 1: free macrophages in bronchiolus and alveoli are strongly positive for ED1 (magnification x 200).
Ta).
AW
Figure 5. Lymph node medulla, 1 pm plastic section after pre-embedding staining with EDi (a) and ED3 (b) in an
immunoperoxidase method, counterstained with toluidin blue (magnification x 320). (a) EDi recognizes an intracellularly
located antigen (arrow). (b) ED3 shows membrane staining of the medullary macrophages (arrow).
596Heterogeneity of lymphoid mononuclear phagocytes
Table 2. Percentages of monocytes/macrophages (as judged
by their morphology and acid phosphatase activity) positive
for either of the three monoclonal antibodies
EDI ED2 ED3 Total M0*
Spleen 87% 90% 26% 9%
Mesenteric lymph node 80% 25% 50% 5%
Thymus 83% 72% 5% 3%
Peripheral blood 90% - - 5%
Bone marrow 82% 72% - 9%
Peritoneal cavity (resident) 98% 45% 5% 70%
Veiled (dendritic) cells 98% - - -
* Total number of monocytes/macrophages (percentage
of nucleated cells) per organ.
antibodies recognize macrophages is assessed by morphological criteria, the tissue distribution and the
correlation with acid phosphatase activity. In addition, it has been demonstrated that the spleen cells,
positive for any of the three antibodies, are capable of
phagocytosis. The monoclonal antibodies did not
react with other cell types or tissue structures.
In the compartments of the lymphoid organs,
distinct macrophage subpopulations can be recognized exclusively by use of the monoclonal antibodies.
EDl showed a patchy staining pattern in all compartments of the organs investigated, and recognized
most macrophage subpopulations as well as monocytes in the peripheral blood. The majority of both
tissue macrophages and free macrophages (alveolar
and peritoneal macrophages) are positive for ED 1.
ED2 recognizes a membrane antigen of most tissue
macrophages, e.g. in connective tissue in all organs
tested, the splenic red pulp, the liver (cells of Von
Kupffer) as well as a part of the peritoneal macrophages. In the thymus, ED2 stained the cortical
macrophages, whereas the medulla was completely
negative. The extensive cell processes of the
ED2+ED3- cells in intimate contact with the surrounding lymphocytes and the exclusive localization
in the cortex might indicate a possible role in the
maturation of cortical thymocytes, as suggested by
Beller & Unanue (1978). Also, in bone marrow, large
ED2+ED3- cells were found within islands of erythropoietic cells; these central macrophages are thought
to have regulatory functions with respect to haemopoiesis (Calderon & Unanue, 1975; Shaklai & Tavassoli, 1979). In peripheral lymphoid organs, only very
few cells stained with ED2.
ED3+ED2- cells form a particular subpopulation
of macrophages, in the splenic white pulp consisting of
the marginal zone macrophages (Humphrey & Grennan, 1981) and the marginal metallophils (Snook,
1964), and in the lymph node consisting of the
subsinusoidal macrophages and the major portion of
the medullary macrophages. This distribution resembles the localization of FITC Ficoll after injection, by
which a distinct macrophage subpopulation can be
distinguished in mice (Humphrey & Grennan, 1981).
Taken together, these results present evidence that
the phenotype of macrophages differs depending on
the organ localization. EDl recognizes a cytoplasmic
antigen, distributed more or less randomly in all
compartments of the lymphoid organs. ED2 and ED3
recognize membrane antigens, by which separate
subpopulations of macrophages can be distinguished
in lymphoid compartments. On the other hand, there
exists a considerable overlap between the distribution
of these two antigens. These results suggest that ED2
and ED3 may recognize differentiation antigens of
macrophages.
The tissue distribution of ED1, ED2 or ED3 does
not correlate with that of any of the macrophageantigens described in mice. Neither of the Mac-1,
Mac-2 and Mac-3 antigens are located exclusively on
macrophages (Flotte et al., 1982). By the localization
pattern of Mac-1, Mac-2 and Mac-3, subpopulations
ofmacrophages can also be distinguished (Flotte et al.,
1982), but none of these correlates exactly with one of
the subpopulations as recognized by ED1, ED2 or
ED3. F4/80 antigen is located exclusively on macrophages in mice, with a distribution pattern comparable
with ED2 (Hume et al., 1983) but, in contrast with
ED2, F4/80 is present in both thymic medulla and
cortex. W3/25 monoclonal antibody recognizes macrophages in rat, but T-helper cells are also positive
(Barclay, 198 lb).
Many of the cells recognized by ED2 and ED3 have
a dendritic appearance. This makes it necessary to
compare them with the other dendritic cells described
in lymphoid organs, i.e. the follicular dendritic cell
(Nossal et al., 1968 and the interdigitating cell. None
of the antibodies react with the FDC, which is not
surprising since considerable evidence exists to suggest
that the FDC originates from a reticulum cell (Veerman, 1975; Villena et al., 1983; Dijkstra, Kamperdijk
& Dopp, 1984), and not from a member of the
mononuclear phagocyte system. There are, however,
studies that describe macrophage-related antigens on
FDCs (Gerdes et al., 1983).
The interdigitating cells (IDCs) occur in the T-cell
areas of lymphoid organs and can be recognized on
597598 C. D. Dijkstra et al.
ultrastructural level (Veldman, 1979; Veerman, 1974)
or by staining for Ia-antigen. Staining with anti-Ia
antibody shows a considerable number of strongly
positive cells with a dendritic appearance (IDCs) in all
T-cell compartments of lymphoid organs (HoffmanFezer et al., 1978; Barclay, 1981a; Dijkstra, 1982). In
all the T-cell areas investigated, EDI showed a patchy
staining pattern, the distribution of which was similar
to that of Ia-positive cells. Both Ia-positive and
ED1-positive cells in T-cell areas showed acid phosphatase activity. Dendritic cells (Steinman & Nussenzweig, 1980) or veiled cells (Knight et al., 1983) isolated
from lymphoid organs, are thought to be in vitro
equivalents of IDCs (Dijkstra, 1982). In cell suspensions enriched for veiled cells (Knight et al., 1983), cells
with characteristics of dendritic cells are positive for
EDI and negative for ED2 and ED3. Together, these
results indicate that interdigitating cells in vivo and
dendritic cells in vitro are both positive for ED 1,
confirming the suggestion that these cells are closely
related and both belong to the mononuclear phagocyte system (Veerman, 1974).
The three monoclonal antibodies described in this
study have considerable value in the identification of
rat macrophages. In particular, ED2 and ED3 enable
us to elucidate the functional aspects and differentiation pathways of the macrophage subpopulations
recognized by these unique antibodies.
ACKNOWLEDGMENTS
The authors wish to thank B. W. C. van der Ven and
W. M. J. M. Bogers for their contribution to this work.
REFERENCES
BARCLAY A.N. (1981a) Different reticular cells in rat lymphoid tissues identified by localization of Ia, Thy-l and
MRC-Ox2 antigen. Immunology, 44, 727.
BARCLAY A.N. (1981b) The localization of populations of
lymphocytes defined by monoclonal antibodies in rat
lymphoid tissues. Immunology, 42, 593.
BEELEN R.H.J., BROEKHUIs-FLUITSMA D.M. & HOEFSMIT
E.C.M. (1978) Peroxidatic activity in mononuclear
phagocytes. Ultramicroscopy, 3, 129.
BELLER D.I. & UNANUE E.R. (1978) Thymic macrophages
modulate one stage of T-cell differentiation in vitro. J.
Immunol. 121, 1861.
BURSUKER I. & GOLDMAN R. (1983) On the origin of
macrophage heterogeneity: a hypothesis. J. Reticuloendothel. Soc. 33, 207.
CALDERON J. & UNANUE E.R. (1975) Two biological activities
regulating cell proliferation in cultures of peritoneal
exudate cells. Nature (Lond.), 253, 359.
COHN Z.A. (1978) The activation of mononuclear phagocytes: fact, fancy and future. J. Immunol. 121, 813.
COHN Z.A. & BENSON B. (1964) The differentiation of
mononuclear phagocytes. Morphology, cytochemistry
and biochemistry. J. exp. Med. 121, 153.
DIJKSTRA C.D. (1982) Characterization of non-lymphoid
cells in rat spleen with special reference to strongly
Ia-positive branched cells in T-cell areas. J. Reticuloendothel. Soc. 32, 167.
DIJKSTRA C.D., KAMPERDIJK E.W.A. & DoPP E.A. (1984)
The ontogenetic development of the follicular dendritic
cell. An ultrastructural study by means of intravenously
injected horseradish peroxidase (HRP)-anti-HRP complexes as marker. Cell Tissue Res. 236, 203.
DUIJVESTJN A.M., SMINIA T., KOHLER Y.G., JANSE E.M. &
HOEFSMIT E.C.M. (1982) The rat thymus microenvironment: a morphological and functional characterization.
In: In Vivo Immunology (eds P. Nieuwenhuis, A. A. Van
den Broek and M. G. Hanna), p. 441. Plenum Press, New
York.
FAZEKAS DE ST GROTH S. & SCHEIDEGGER D. (1980) Production of monoclonal antibodies: stratology and tactics. J.
immunol. Meth. 35, 1.
FLOTTE T.J., SPRINGER T.A. & THORBECKE G.J. (1982)
Dendritic cell and macrophage staining by monoclonal
antibodies in tissue sections and epidermal sheets. Am. J.
Pathol. 111, 112.
GERDES J., STEIN H., MASON D.Y. & ZIEGLER A. (1983)
Human dendritic reticulum cells of lymphoid follicles:
their antigenic profile and their identification as multinucleated giant cells. Virch. Arch. 42, 161.
HAINES K.A., FLOTrE T.J., SPRINGER T.A., GIGLI I. &
THORBECKE G.J. (1983) Staining of Langerhans cells with
monoclonal antibodies to macrophages and lymphoid
cells. Proc. natn. Acad. Sci. U.S.A. 80, 3448.
HOEFSMIT E.C.M., KAMPERDIJK E.W.A., HENDRIKS H.R.,
BEELEN R.H.J. & BALFOUR B.M. (1980) Lymph node
macrophages. In: The Reticuloendothelial System (eds I.
Carr and W. T. Daems), Vol. I, p.417. Plenum Publishing
Corporation, New York.
HOFFMAN-FEzER G., GOTZE D., RODT H. & THIERFELDER S.
(1978) Immunohistochemical localization of xenogeneic
antibodies against lak lymphocytes on B cells and
reticular cells. Immunogenetics, 6, 367.
HUME D.A., ROBINSON A.P., MACPHERSON G.G. & GORDON
S. (1983) The mononuclear phagocyte system of the
mouse defined by immunohistochemical localization of
antigen F4/80. J. exp. Med. 158, 1522.
HUMPHREY J.H. & GRENNAN D. (1981) Different macrophage
populations distinguished by means of fluorescent polysaccharides. Recognition and properties ofmarginal zone
macrophages. Eur. J. Immunol. 11, 221.
KNIGHT S.C., MERTIN J., STACKPOOLE A. & CLARK J. (1983)
Induction of immune responses in vivo with small
numbers of veiled (dendritic) cells. Proc. natn. Acad.
U.S.A. 80, 6032.
KOHLER G. & MILSTEIN C. (1975) Continuous cultures of
fused cells secreting antibody of defined specificity.
Nature (Lond.), 256. 495.Heterogeneity of lymphoid mononuclear phagocytes 599
MORAHAN P.S. (1980) Macrophage nomenclature: where are
we going? J. Reticuloendothel. Soc. 27, 223.
NOSSAL G.J.V., ABBOT A., MITCHELL J. & LUMMUS Z. (1968)
Antigens in immunity. XV. Ultrastructural features of
antigen capture in primary and secondary follicles. J. exp.
Med. 127, 227.
PEARCE A.G.E. (1968) Histochemistry. Theoretical and
Applied. Vol. 1, 3rd edn. Churchill Livingstone, Edinburgh.
SHAKLAI M. & TAVASSOLI M. (1979) Cellular relationship in
the rat bone marrow studied by freeze fracture and
lanthanum impregnation thin-sectioning electron microscopy. J. Ultrastruc. Res. 69, 343.
SNOOK T. (1964) Studies on the perifollicular region of the
rat's spleen. Anat. Rec. 148, 149.
SORG C. (1982) Heterogeneity of macrophages in response to
lymphokines and other signals. Molec. Immunol. 19,
1275.
STEINMAN R.M. & NUSSENZWEIG M.C. (1980) Dendritic cells:
features and functions. Immunol. Rev. 53, 127.
STREEFKERK J.G. & VAN DER PLOEG M. (1974) The effect of
methanol on granulocyte and horseradish peroxidase.
Quantitatively studied in a film model system. Histochemistry, 40, 105.
TWOMEY J.J., LAUGHTER A. & BROWN M.F. (1983) A
comparison of the regulatory effects of human monocytes, pulmonary alveolar macrophages (PAMs) and
spleen macrophages upon lymphocyte responses. Clin.
exp. Immunol. 52, 449.
VAN FURTH R., COHN Z., HIRSCH J.G., SPECTOR W.G. &
LANGEVOORT H.L. (1972) The mononuclear phagocyte
system: a new classification of macrophages, monocytes
and their precursor cells. Bull. Wid Hlth Org. 46, 845.
VEERMAN A.J.P. (1974) On the interdigitating cells in thymusdependent area of the rat spleen: a relation between the
mononuclear phagocyte system and T lymphocytes. Cell
Tissue Res. 148, 247.
VEERMAN A.J.P. (1974) On the interdigitating cells in the
thymus-dependent area of the rat spleen: a relation
between the mononuclear phagocyte system and T lymphocytes. Cell Tissue Res. 148, 247.
VELDMAN J.E. (1970) Histophysiology and electron microscopy of the immune response. Ph.D Thesis. Groningen,
The Netherlands.
VILLENA A., ZAPATA A., RIVERA-POMAR J.M., BARRUTIA
M.G. & FONFRIA J. (1983) Structure of the non-lymphoid
cells during the postnatal development of the rat lymph
nodes, fibroblastic reticulum cells and interdigitating
cells. Cell Tissue Res. 229, 219.
WALKER W.S. (1976) Functional heterogeneity of macrophages in the induction and expression of acquired
immunity. J. Reticuloendothel. Soc. 20, 57.