Immunology 1990 70 513-519
A monoclonal antibody, RbM2, specific for a lysosomal membrane antigen of rabbit monocyte/macrophages Y. SHIMOKAWA,*t M. TAKEYAt Y. MIYAUCHI* & K. TAKAHASHIt *First Department of Surgery and tSecond Department of Pathology, Kumamoto University Medical School, Kumamoto, Japan Acceptedfor publication 20 April 1990
SUMMARY An anti-macrophage monoclonal antibody (mAb), RbM2, was produced using thioglycolate-elicited rabbit peritoneal macrophages as immunogen. The immunohistochemical approach revealed an intense reactivity of this mAb to macrophages, particularly those engaged in phagocytosis, in different organs and tissues, and to peripheral blood monocytes. Immunoelectron microscopy demonstrated reaction products of RbM2 in the lysosomes of macrophages and monocytes. This selective reactivity with the mAb was further confirmed in various experiments by endocytosis of Latex particles with different diameters or IgG-coated sheep erythrocytes. The results indicate that RbM2 recognizes a lysosomal membrane antigen of 50,000 molecular weight (MW). In contrast, dendritic cells, such as follicular dendritic cells (FDC) of lymphoid follicles, interdigitating cells (IDC) of lymphoid T zones, or epidermal Langerhan's cells, were not reactive with the antibody. Thus, RbM2 is useful not only in differentiating the phagocytic macrophages from the immunologically accessory dendritic cell populations but also in identifying lysosomes and their related structures in macrophages.
INTRODUCTION Monoclonal antibodies (mAb) against markers for cell population, differentiation and activation of monocyte/macrophages or dendritic cells have been produced in humans, mice, rats, guinea-pigs and rabbits (Anderson et al., 1986; Parwaresch et al., 1983; Austyn & Gordon, 1981; Flotte, Springer & Thorbecke, 1983; Takeya et al., 1989; Takeya, Hsiao & Takahashi, 1987; Kraal et al., 1988; Tsukada et al., 1986; Watanabe et al., 1985). They are useful tools in discriminating various cell populations based on differences in tissue localization, cell morphology, differentiation or activation. The mAb against macrophages or their related cells are known to recognize various subcellular components; cell membranes (Austyn & Gordon, 1981; Takeya et al., 1987), lysosomal granules (Chen et al., 1985; Mane et al., 1989), phagocytic vacuoles (Kreipe et al., 1987; Parwaresch et al., 1986; Radzun et al., 1987), filaments (Ukai et al., 1988), or cytosolic components (Tsukada et al., 1986; Ukai et al., 1988). Among these, a few mAb which recognize lysosomal components of macrophages have been reported in mice and humans (Chen et al., 1985; Mane et al., 1989). In rabbits, two mAb against macrophages have been Abbreviations: DAB, diaminobenzidine, FDC, follicular dendritic cells; IDC, interdigitating cells; PBS, phosphate-buffered saline. Correspondence: Dr K. Takahashi, 2nd Dept. of Pathology, Kumamoto University Medical School, 2-2-1 Honjo, Kumamoto 860, Japan.
513
reported, but neither of them recognizes lysosomal components (Tsukada et al., 1986; Watanabe et al., 1985). The present study describes an anti-rabbit macrophage mAb, termed RbM2, that recognizes the bulk of lysosomal membranes of cells of the monocyte/macrophage population but not those of dendritic cells.
MATERIALS AND METHODS Animals and tissues New Zealand white rabbits and BALB/c mice were obtained from the Shizuoka Agricultural Cooperative Association for Laboratory Animals (Shizuoka) and kept under routine laboratory conditions. To test interspecies reactivities of the antibodies, lymph nodes and spleens were obtained from C3H mice, Wistar rats, guinea-pigs of the Hartley strain, golden hamsters and goats. Human lymph nodes and spleens were harvested from human autopsy cases of non-malignant diseases. Monoclonal antibody production Peritoneal exudate cells were obtained from the rabbits 4 days after intraperitoneal (i.p.) injection of 50 ml of thioglycolate broth (Nissui, Tokyo) and used as immunogen. BALB/c mice were immunized three times by i.p. injection of the rabbit peritoneal exudate cells at 2-week intervals. Four days after the last immunization, mouse spleen cells were collected, and 3 x 108 of the spleen cells were fused with 1 5 x I07 of NS-l mouse myeloma cells by polyethyleneglycol 4000 (Merck, Darmstadt, FRG), as described previously (Kohler & Milstein, 1975).
514
Y. Shimokawa et al.
Hybrids were selected with HAT medium (hypoxanthine l X 10-4 M, aminopterine 4 x I0-7 M and thymidine l-6 x 10-5 M; Sigma, St Louis, MO). Screening of the mAb-producing hybrids was performed on cryostat sections of rabbit spleens by the twostep immunoperoxidase method described below. Clones producing antibodies reactive with splenic macrophages were selected, cloned twice by the limiting dilution procedure, and processed for mass culture. Ascites was obtained by injection of 5 x 106 hybrid cells into pristane-primed BALB/c mice. Determination of isotype The isotype of RbM2 was determined by the Ouchterlony method using a monoclonal typing kit (Miles, Naperville, IL).
Immunohistochemistry After killing with pentobarbital anaesthesia, the following tissues were excised from the rabbits and fixed with 2% periodate-lysine-paraformaldehyde (McLean & Nakane, 1974) for 4 hr: spleen, lymph nodes, thymus, liver, lungs, heart, aorta, stomach, Peyer's patches, kidneys, testes, ovaries, uterus, skeletal muscles, salivary glands, skin and brain. After washing in phosphate-buffered saline (PBS) containing a graded series of sucrose (10%, 15%, 20%), the tissues were rinsed for 1 hr in PBS containing 20% sucrose and 5% glycerol, then embedded in tissue embedding compound, and frozen in dry-ice acetone. Cryostat sections were cut at 6-8 gm thick and dried in air. The omentum was spread on albumin-coated slides without sectioning. Peritoneal macrophages and peripheral blood cells were prepared as cell smears and fixed in acetone. Immunostaining was performed as described previously (Takeya et al., 1987). In brief, the cryostat sections, spread omentum and cell smears were pretreated with 5 mm periodate for 10 min, washed with PBS, and immersed in 3 mm sodium borohydride for 30 min, according to the method of Isobe et al. (1977), to inhibit endogenous peroxidase activity. The specimens were incubated with culture supernatant or ascites diluted 1: 1000 in PBS as primary antibody for 1 hr, washed well with PBS, and covered with species-specific sheep anti-mouse Ig [F(ab')2] conjugated with peroxidase (Amersham, Amersham, Bucks, U.K.) as secondary antibody for 1 hr. To visualize peroxidase activity, the specimens were incubated with 3,3'-diaminobenzidine (DAB) H202 as substrate. Counter staining was done with haematoxylin for tissue specimens and with the Giemsa method for smears. As the control, tissue specimens were incubated with non-immunized mouse serum or PBS instead of primary antibody, and then processed by the same procedure described above. Tissues of species other than mouse were processed and stained in the same manner as for rabbit tissues. In immunostaining mouse tissues, biotinylated primary antibodies were applied first, followed by treatment with avidin-biotin-peroxidase complex. Visualization of peroxidase activity was performed as mentioned above. Immunoelectron microscopy Rabbit peritoneal exudate cells obtained as described above were incubated on Petri dishes with RPMI-1640 supplemented with 10% fetal calf serum at 370 for 30 min. After removing the non-adherent cells, the adherent cells were fixed with 0 05% glutaraldehyde in 0-165 M cacodylate buffer, pH 7-4, for 10 min. Immunostaining was performed as described above, with minor
modification. Incubation with the mAb was performed overnight, and that with the second antibody was done for 3 hr. After detection of peroxidase activity with DAB, the cells were postfixed with 1% osmium tetroxide for 1 hr, dehydrated through a graded series of ethanols, and detached from the dishes by adding propylene oxide. The cell pellets were embedded in Epon 128 and sectioned by an Ultrotome Nova (LKB, Uppsala, Sweden). To collect peripheral blood monocytes, blood was obtained by cardiac puncture from the rabbits under pentobarbital anaesthesia and decoagulated with heparin. Collected blood was diluted two-fold with RPMI-1640. After gently adding Lymphoprep (Nycomed AS, Oslo, Norway), the diluted blood was centrifuged at 400 g for 30 min to separate the mononuclear cell layer. This cell layer was fixed, immunostained, and processed for electron microscopy in the same manner mentioned above. The sections were observed under an electron microscope H-300 (Hitachi, Tokyo) without poststaining. Enzyme cytochemistry For subcellular demonstration of acid phosphatase, adherent peritoneal macrophages obtained as described above were fixed in 2 5% glutaraldehyde for 1 hr and then subjected to the Gomori method as modified by Barka & Anderson (1963). Latex particle endocytosis Adherent peritoneal macrophages obtained as above were incubated for 30 min at 0°, 16° and 370 after the addition of Latex particles with different diameters (0-1 ,um and 0-8 ,um; Sigma) and submitted to immunoelectron microscopy as described above. To inhibit phagocytosis, adherent cells were incubated 30 min at 370 in the culture medium containing 20 ,ug/ ml of Cytochalasin B (Sigma) and processed for immunoelectron microscopy. Immune phagocytosis Adherent peritoneal macrophages were incubated for 1 hr at 370 or below 160 in the medium containing the same volume of sheep erythrocytes (E) coated with anti-E antibody (IgG) (IgG-EA) adjusted to 1 x 108/ml. They were then fixed as described above and subjected to immunoelectron microscopy. Estimation of molecular weight To estimate the molecular weight (MW) of RbM2-recognized antigen, proteins were extracted from rabbit peritoneal macrophages with 1% n-octyl-fl-glucoside in phosphate buffer, dissolved in sample buffer [0-5 M Tris-HCl buffer 2 5 ml, 10% sodium dodecyl sulphate (SDS) 4 ml, glycerin 2 ml, 2-mercaptoethanol 1 ml, 0 05% bromophenol blue 0 54 ml and distilled water 10 ml], and boiled for 4 min. Using SDS-PAGE mini (Tefco, Tokyo), electrophoresis was performed on an 8% SDSPAGE gel. Proteins were blotted for I hr at 40 on nitrocellulose membranes (Millipore, Bedford, MA). Membranes were immunostained using the blotting detection kit (Amersham). Briefly, after the membranes were blocked with 10% dried milk solution, they were incubated with primary antibody solution for I hr at room temperature, followed by incubations with a biotinylated anti-mouse immunoglobulin antibody for 20 min and then with a streptavidin-biotinylated alkaline phosphatase complex for 20 min. Alkaline phosphatase activity was visualized by incubating the membranes with substrate solution that
Monoclonal antibody RbM2 had been made by adding a drop of nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate solutions into each 10 ml of diethanolamine buffer. Whole protein patterns of rabbit peritoneal macrophages and a molecular weight standard were stained with Amido black. RESULTS Isotype of the mAb On the frozen sections, three out of 35 hybridomas were found to be producing antibodies reactive with splenic macrophages. In the present paper, one of them was chosen, designated RbM2, and further characterized. The isotype of RbM2 was IgG 1.
Distribution, morphology and immunohistochemical characterization of RbM2-reactive cells The mAb RbM2 was found to react exclusively with phagocytes in different organs and tissues, as determined by immunohistochemistry. The cells were defined as macrophages based on their morphology and localization in the various compartments of the lymphoid and non-lymphoid organs. In general, the larger and more phagocytic the macrophages became, the more intensely they stained with the antibody. At high magnification, intense reactivity was observed in the cytoplasmic granules of phagocytosing macrophages. In the red pulp of the spleen, RbM2 reacted intensely with cordal macrophages, which were large and round or polygonal (Fig. la). In the white pulp, a few polygonal macrophages were reactive in the germinal centre and mantle zone, but there was no staining of marginal metallophils, IDC in the periarteriolar lymphatic sheaths (PALS), or FDC in the germinal centre. In the lymph nodes, the reaction for RbM2 was limited to sinus macrophages alone, particularly those in the medullary sinuses, and to macrophages in the lymphoid follicles (Fig. Ib). In the lymph sinuses, RbM2-reactive macrophages were large and mostly polygonal. In the lymphoid follicles, tingible body macrophages also stained positively. However, no reactivity was detected in any dendritic cells, including IDC in the paracortical areas and FDC in the germinal centre. In the thymus, polygonal macrophages reactive with RbM2 distributed sporadically (Fig. lc), but thymic epithelia or dendritic cells showed no reactivity. In the Peyer's patches, intense reactivity was detected in round or polygonal macrophages in the lymphoid follicles, particularly in the germinal centre. Similar positive cells were found in the dome of lymphoid follicles and interfollicular areas (Fig. I d), but cells with dendritic morphology did not stain. In the liver, Kupffer cells reacted with RbM2, but not with the sinusoidal endothelia, perisinusoidal fat-storing cells or parenchymal cells (Fig. Ie). In the lungs, alveolar macrophages showed a positive reaction for the antibody (Fig. lf), and a few interstitial macrophages were also reactive with it. In the skin, polygonal or spindle dermal macrophages were stained positively, but no reactivity was detected in any epidermal dendritic cells, including Langerhan's cells. In the brain, a positive reaction for RbM2 was demonstrated in round or polygonal macrophages in the subarachnoid space and in some perivascular spindle cells in the perivascular space, but resting microglia were unreactive.
515
Peritoneal macrophages reacted with RbM2 (Fig. Ig, h) and intense reactivity was found in their cytoplasmic granules. However, their cytoplasm was often stained diffusely due to the diffusion of immunoreactive products for RbM2 from the cytoplasmic granules into the cytoplasm, which occurs in immunostaining, probably by visualization of peroxidase activity. In the omentum, RbM2-reactive macrophages mostly appeared round or polygonal (Fig. Ig), but a few were spindle. In the milky spots, a large number of variable-sized, round cells accumulated, mostly reacting with the antibody. Further, RbM2-positive macrophages were dispersed in the subcutaneous tissues, the mucosa of the gastrointestinal tract, the endometrium, and the interstitial tissues of different organs and tissues. Besides, monocytes stained positively with RbM2. Polymorphonuclear leucocytes showed no reactivity, and lymphocytes, erythrocytes or other kinds of cells, including epithelial, endothelial, neuronal and muscle cells, did not stain with the antibody. These results indicate that this mAb reacts with the monocyte/macrophage population alone and not with dendritic cells. Over 90% of the peritoneal macrophages in ascites and the monocytes in the peripheral blood also reacted with RbM2. However, it was not reactive with the macrophages of mice, rats, guinea-pigs, hamsters, goats or humans. Intracellular localization of reactivity with RbM2 in macrophages and monocytes Immunoelectron microscopy revealed that reaction products of RbM2 were confined to variable-sized vacuoles of peritoneal macrophages (Fig. 2a), which were consistent with the lightmicroscopic immunohistochemical findings mentioned above. RbM2-reactive vacuoles were mostly large and irregular, and some contained certain digested or degraded materials, suggesting secondary lysosomes. Although reactivity was also found around some large vacuoles, it was considered to be an artifact caused by diffusion of the reagent during immunostaining. Reactivity with the antibody was also detected in primary lysosomes, but pinocytic vesicles, tubular structures and endosomes (receptosomes), with or without multivesicular profiles, were not reactive with the mAb. In addition, the cell membrane, nuclear envelope and intracellular organelles other than lysosomes showed no reactivity. In monocytes, the localization pattern of RbM2 was essentially the same. Ultrastructural cytochemistry of acid phosphatase activity revealed a localization pattern similar to that of the reactivity with RbM2 (Fig. 2b). This result indicates that this mAb recognizes lysosomes and their related compartments. In vitro investigation of the reactivity with RbM2 in macrophages during endocytosis On incubation at 00 for 30 min in the medium containing Latex particles with diameters of 0-8 gm and 0l 1 m, no specific binding of these particles was found on the cell surface of peritoneal macrophages. On temperature shifting to 370, they started endocytosing Latex particles in the fluid phase. At 5 min after temperature shifting, the macrophages ingested many Latex particles, around most of which the reaction products of RbM2 were demonstrated immunoelectron microscopically (Fig. 3a). However, when incubated in the same medium below
516
r_E1 .^_:.
Y. Shimokawa et al.
g
.I
._
_
_
:l.5+:** :.
*4
_ t
*
*:_
|
.uS
::n..
:^s
sw
w
,'!:
t
I_
._a
E
t
t
,,,_
.. ^
^:: _
..
#
.:
_
,_
w
Figure 1. Distribution of RbM2-positive cells in various organs and tissues of normal rabbits. (a) Macrophages in the red pulp (rp) and white pulp (wp) of the spleen ( x 180). (b) Macrophages in the medullary sinuses (ms), and tingible body macrophages in the lymph follicle (fo) of the lymph node ( x 160). (c) Macrophages in the thymic cortex and medulla ( x 110). (d) Macrophages in the lymph follicles and interfollicular regions of the Peyer's patch (x 70). (e) Kupffer cells in the hepatic sinusoids (x 180). (f) Alveolar macrophages (x 400). (g) Macrophages in the milky spot (ms) of the omentum (x 110). (h) Peritoneal macrophages (arrow); lymphocytes and granulocytes are negative (arrowhead) (x 540). Staining with two-step immunoperoxidase method using RbM2.
517
Monoclonal antibody RbM2 DISCUSSION
Figure 2. (a) Peritoneal macrophages exhibit a positive reaction with RbM2 in phagocytic vacuoles and primary lysosomes (arrow). Immunoelectron microscopy with RbM2 (bar= 1 ,m). (b) In the macrophages, phagocytic vacuoles (heterolysosomes) and primary lysosomes (arrow) are positive for acid phosphatase. Barka & Anderson (1963) method (bar= 1 ,m).
16", the temperature known to inhibit lysosomal fusion, the ingestion of Latex particles by the cells decreased, and no reactivity with the mAb was detected in any vesicles or vacuoles containing the particles (Fig. 3b). These results indicate that among Latex particles internalized by the macrophages via fluid phase endocytosis, RbM2 recognizes only those fused with lysosomes. When cultured in the medium to which Cytochalasin B was added to inhibit phagocytosis, the macrophages ingested only the Latex particles with a diameter of 0 1 ,um, not those of 0-8 ,m. With time, the Latex particles were pinocytosed as a single particle, aggregated and fused with each other into large phagocytic vacuoles. RbM2 reacted with such large vacuoles but not with the pinocytic vesicles containing a single particle (Fig. 3c). Cytochemically, acid phosphatase activity was demonstrated in these large phagocytic vacuoles alone. This indicates that they are heterolysosomes. In the experiment of immune phagocytosis with IgG-EA, reactivity with RbM2 was detected around sheep erythrocytes phagocytosed by macrophages (Fig. 3d). MW of RbM2-recognized antigen
RbM2 immunostained a protein band of 50,000 MW in comparison with the MW standard stained with Amido black (Fig. 4).
The mAb RbM2 reacts with rabbit macrophages, as determined by tissue distribution and cell morphology. It is also reactive with peripheral blood monocytes, but not with granulocytes or other blood cells. Dendritic accessory cells of the T- or B-cell immune response, such as IDC in the lymphoid T zone, epidermal Langerhan's cells, or FDC within the lymph follicles, show no reactivity with RbM2. RbM2 showed no cross-reaction to macrophages in lymph nodes of humans and animals other than rabbits. Thus, RbM2 is a suitable reagent to differentiate the monocyte/macrophage population from the dendritic accessory cell populations in rabbits. In previous studies, two anti-rabbit macrophage mAb were produced, which were termed SRM 1 and RAM 11 (Watanabe et al., 1985; Tsukada et al., 1986). Compared to RbM2, they show a nearly identical reactivity with macrophages in distribution, but they differ from each other in isotype, recognition site and kind of immunoreactive cells other than macrophages. Our approach by immunoelectron microscopy demonstrated that RbM2 is confined to phagocytic vacuoles or to cytoplasmic granules in the monocyte/macrophage population. Ultrastructural cytochemistry for the demonstration of acid phosphatase revealed that RbM2-positive phagocytic vacuoles or cytoplasmic granules are positive for acid phosphatase, thus indicating that they are consistent with secondary or primary lysosomes. Near the cell membrane, we found some vacuoles or vesicles unreactive with RbM2 and showing no positive reaction for acid phosphatase activity. These vacuoles or vesicles are considered to be endocytic vesicles or vacuoles prior to fusion with lysosomes. To demonstrate the recognition site of RbM2 more clearly, we performed various experiments of endocytosis by rabbit peritoneal macrophages and monocytes. Endocytosis is discriminated into phagocytosis and pinocytosis depending on the size of the particles ingested, and phagocytosis is defined as the internalization of large particles greater than 0 1 ,um, a process known to be inhibited by Cytochalasin B (Wills et al., 1972). Based on these facts, we examined the immunoreactivity of RbM2 in the macrophages and monocytes during endocytosis of particles of different sizes. As a result, immunoreactivity with RbM2 in the cells was confirmed to be independent of the type of endocytosis, i.e. phagocytosis or pinocytosis and fluid phase endocytosis or receptor-mediated endocytosis. This was because almost all the heterolysosomes were labelled by RbM2 in the processes of ingestion of variously sized Latex particles and IgG-sensitized erythrocytes. In all these endocytosis experiments, there was no immunoreactivity with RbM2 around the particles at temperatures below 16°, but on temperature shift to 370 there was. These data indicate that RbM2 recognizes lysosomes and not endosomes or endocytic vesicles. In secondary lysosomes, immunoreactivity with RbM2 was observed more intensely near the lysosomal membrane, suggesting that this mAb recognizes a lysosomal membrane antigen. From the fact that the mAb does not react with any other kind ofcell, even lysosome-rich cells such as hepatocytes, neuronal cells, or renal tubular epithelia, it is suggested that this antigen is a glycoprotein of 50,000 MW specifically localized on the lysosomal membrane of the monocyte/macrophage population alone. Furthermore, we speculate that the antigen is one of the
Y. Shimokawa et al.
518
aJ4!~~~~~ Figure 3. Immunoreactivity of peritoneal macrophages with RbM2 during endocytosis. (a) On incubation with Latex particles (0 8 ium) for 20 mmn at 370, some particle-containing vacuoles react with RbM2 (arrow), and others are unreactive (arrowhead) (bar = 1 jim). (b) On incubation below 16°, almost all the Latex particles show no reaction against RbM2 (bar = 1 tim). (c) On incubation at 370 after adding Latex particles (0-1 jim) to the medium, large phagocytic vacuoles containing many particles are reactive with RbM2, and single particle-containing endosomes are unreactive (bar = 1 jim) (arrows). (d) Incubation with sensitized sheep erythrocytes at 370 for 20 min. Two erythrocytes are ingested by the macrophage: one is a heterolysosome reacting with RbM2, the other is unreactive during the endosomal stage (arrow) (bar = 1 jim). Immunoelectron microscopy with RbM2.
(a)
50,000
(b) (c) .MW _ 1111
E
I97400 -|966,200 42eOO
I 421,500 4
14,400
Figure 4. Molecular weight of an antigen recognized by RbM2. (a) RbM2 immunostaining. (b) Whole protein pattern of peritoneal macrophages. (c) Molecular weight standard.
essential components of the lysosomal membrane and is increased in secondary lysosomes, because RbM2 is expressed in not only secondary lysosomes but also primary lysosomes, more prominently in heterolysosomes (secondary lysosomes), and irrespective of type of endocytosis. To elucidate the property of the lysosomal membrane antigen, biochemical and immunological investigations are required.
ACKNOWLEDGMENTS This work was done at the Second Department of Pathology, Kumamoto University Medical School, and supported in part by a grant-inaid for scientific research from the Ministry of Education, Welfare and
Culture, Japan.
REFERENCES ANDERSON C.L., GuYRE P.M., WHITIN J.C., RYAN D.H., LOONEY R.J. & FANGER M.W. (1986) Monoclonal antibodies to Fc receptors for IgG on human mononuclear phagocytes: antibody characterization and induction of superoxide production in a monocyte cell line. J. biol.
Chem. 261, 12856. AUSTYN J.M. & GORDON S. (1981) F4/80, a monoclonal antibody directed specifically against the mouse macrophage. Eur. J. Immunol. 11, 805. BARKA T. & ANDERSON P.J. (1963) Histochemistry: Theory, Practice, and Bibliography, p. 239. Harper & Row, New York. CHEN J.W., MURPHY T.L., WILLINGHAM M.C., PASTAN I. & AUGUST J.T. (1985) Identification of two lysosomal membrane glycoproteins. J. Cell Biol. 101, 85. FLoTTE T.J., SPRINGER T.A. & THORBECKE G.J. (1983) Dendritic cell and macrophage staining by monoclonal antibodies in tissue sections and epidermal sheets. Am. J. Pathol. 111, 112. ISOBE Y., CHEN S.T., NAKANE P.K. & BROWN W.R. (1977) Studies on translocation of immunoglobulins across intestinal epithelium. I. Improvements in the peroxidase-labelled antibody method for application to study of human intestinal mucosa. Acta Histochem. Cytochem. 10, 161. KOHLER G. & MILSTEIN C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature (Lond.), 256,495. KRAAL G., SHIAMATEY-KOOLMA R., HOFFER M., BARKER D. & SCHEPER R. (1988) Histochemical identification of guinea-pig macrophages by monoclonal antibody MR-1. Immunology, 65, 523. KREIPE H., RADZUN H.J., PARWARESCH M.R., HAISLIP A. & HANSMANN M.L. (1987) Ki-M7 monoclonal antibody specific for myelomonocytic cell lineage and macrophages in human. J. Histochem. Cytochem. 35, 1117.
Monoclonal antibody RbM2 MCLEAN I.W. & NAKANE P.K. (1974) Periodate-lysine-paraformaldehyde fixative. A new fixative for immunoelectron microscopy. J. Histochem. Cytochem. 22, 1077. MANE S.M., MARZELLA L., BAINTON D.F., HOLT V.K., CHA Y.,
HILDRETH J.K. & AUGUST J.T. (1989) Purification and characterization of human lysosomal membrane glycoproteins. Arch. Biochem. Biophys. 268, 360. PARWARESCH M.R., RADZUN H.J., HANSMANN M.L. & PETERS K.P.
(1983) Monoclonal antibody Ki-M4 specifically recognizes human dendritic reticulum cells (follicular dendritic cells) and their possible precursor in blood. Blood, 62, 585. PARWARESCH M.R., RADZUN H.J., KREIPE H., HANSMANN M.L. &
BARTH J. (1986) Monocyte/macrophage-reactive monoclonal antibody Ki-M6 recognizes an intracytoplasmic antigen. Am. J. Pathol. 124, 141. RADZUN H.J., KREIPE H., BODEWADT S., HANSMANN M.L., BARTH J. &
PARWARESCH M.R. (1987) Ki-M8 monoclonal antibody reactive with an intracytoplasmic antigen of monocyte/macrophage lineage. Blood, 69, 1320. TAKEYA M., HSIAo L., SHIMOKAWA Y. & TAKAHASHI K. (1989) Heterogeneity of rat macrophages recognized by monoclonal anti-
bodies: An immunohistochemical and immunoelectron microscopic study. J. Histochem. Cytochem. 37, 635.
519
TAKEYA M., HSIAo L. & TAKAHASHI K. (1987) A new monoclonal antibody, TRPM-3, binds specifically to certain rat macrophage populations: Immunohistochemical and immunoelectron microscopic analysis. J. Leukocyte Biol. 41, 187. TSUKADA T., ROSENFELD M., Ross R. & GOWN A.M. (1986) Immunocytochemical analysis of cellular components in atherosclerotic lesions. Use of monoclonal antibodies with the Watanabe and fat-fed rabbit. Arteriosclerosis, 6, 601. UKAi K., TERASHIMA K., Fujii Y. & IMAI Y. (1988) A new monoclonal antibody, UFT-4, reacting with rat Kupffer cells. Immunohistochemical and immunoelectron microscopical analysis with reestimation of the reticuloendothelial system. Acta Pathol. Jpn. 38, 1391. WATANABE T., HIRATA M., YOSHIKAWA Y., NAGAFUCHI Y., ToYOSHIMA H. & WATANABE T. (1985) Role of macrophages in atherosclerosis. Sequential observations ofcholesterol-induced rabbit aortic lesion by the immunoperoxidase technique using monoclonal antimacrophage antibody. Lab. Invest. 53, 80. WILLS E.J., DAVIES P., ALLISON A.C. & HASWELL A.D. (1972) Cytochalasin B fails to inhibit pinocytosis by macrophages. Nature New Biol. 240, 58.
A monoclonal antibody, RbM2, specific for a lysosomal membrane antigen of rabbit monocyte/macrophages Y. SHIMOKAWA,*t M. TAKEYAt Y. MIYAUCHI* & K. TAKAHASHIt *First Department of Surgery and tSecond Department of Pathology, Kumamoto University Medical School, Kumamoto, Japan Acceptedfor publication 20 April 1990
SUMMARY An anti-macrophage monoclonal antibody (mAb), RbM2, was produced using thioglycolate-elicited rabbit peritoneal macrophages as immunogen. The immunohistochemical approach revealed an intense reactivity of this mAb to macrophages, particularly those engaged in phagocytosis, in different organs and tissues, and to peripheral blood monocytes. Immunoelectron microscopy demonstrated reaction products of RbM2 in the lysosomes of macrophages and monocytes. This selective reactivity with the mAb was further confirmed in various experiments by endocytosis of Latex particles with different diameters or IgG-coated sheep erythrocytes. The results indicate that RbM2 recognizes a lysosomal membrane antigen of 50,000 molecular weight (MW). In contrast, dendritic cells, such as follicular dendritic cells (FDC) of lymphoid follicles, interdigitating cells (IDC) of lymphoid T zones, or epidermal Langerhan's cells, were not reactive with the antibody. Thus, RbM2 is useful not only in differentiating the phagocytic macrophages from the immunologically accessory dendritic cell populations but also in identifying lysosomes and their related structures in macrophages.
INTRODUCTION Monoclonal antibodies (mAb) against markers for cell population, differentiation and activation of monocyte/macrophages or dendritic cells have been produced in humans, mice, rats, guinea-pigs and rabbits (Anderson et al., 1986; Parwaresch et al., 1983; Austyn & Gordon, 1981; Flotte, Springer & Thorbecke, 1983; Takeya et al., 1989; Takeya, Hsiao & Takahashi, 1987; Kraal et al., 1988; Tsukada et al., 1986; Watanabe et al., 1985). They are useful tools in discriminating various cell populations based on differences in tissue localization, cell morphology, differentiation or activation. The mAb against macrophages or their related cells are known to recognize various subcellular components; cell membranes (Austyn & Gordon, 1981; Takeya et al., 1987), lysosomal granules (Chen et al., 1985; Mane et al., 1989), phagocytic vacuoles (Kreipe et al., 1987; Parwaresch et al., 1986; Radzun et al., 1987), filaments (Ukai et al., 1988), or cytosolic components (Tsukada et al., 1986; Ukai et al., 1988). Among these, a few mAb which recognize lysosomal components of macrophages have been reported in mice and humans (Chen et al., 1985; Mane et al., 1989). In rabbits, two mAb against macrophages have been Abbreviations: DAB, diaminobenzidine, FDC, follicular dendritic cells; IDC, interdigitating cells; PBS, phosphate-buffered saline. Correspondence: Dr K. Takahashi, 2nd Dept. of Pathology, Kumamoto University Medical School, 2-2-1 Honjo, Kumamoto 860, Japan.
513
reported, but neither of them recognizes lysosomal components (Tsukada et al., 1986; Watanabe et al., 1985). The present study describes an anti-rabbit macrophage mAb, termed RbM2, that recognizes the bulk of lysosomal membranes of cells of the monocyte/macrophage population but not those of dendritic cells.
MATERIALS AND METHODS Animals and tissues New Zealand white rabbits and BALB/c mice were obtained from the Shizuoka Agricultural Cooperative Association for Laboratory Animals (Shizuoka) and kept under routine laboratory conditions. To test interspecies reactivities of the antibodies, lymph nodes and spleens were obtained from C3H mice, Wistar rats, guinea-pigs of the Hartley strain, golden hamsters and goats. Human lymph nodes and spleens were harvested from human autopsy cases of non-malignant diseases. Monoclonal antibody production Peritoneal exudate cells were obtained from the rabbits 4 days after intraperitoneal (i.p.) injection of 50 ml of thioglycolate broth (Nissui, Tokyo) and used as immunogen. BALB/c mice were immunized three times by i.p. injection of the rabbit peritoneal exudate cells at 2-week intervals. Four days after the last immunization, mouse spleen cells were collected, and 3 x 108 of the spleen cells were fused with 1 5 x I07 of NS-l mouse myeloma cells by polyethyleneglycol 4000 (Merck, Darmstadt, FRG), as described previously (Kohler & Milstein, 1975).
514
Y. Shimokawa et al.
Hybrids were selected with HAT medium (hypoxanthine l X 10-4 M, aminopterine 4 x I0-7 M and thymidine l-6 x 10-5 M; Sigma, St Louis, MO). Screening of the mAb-producing hybrids was performed on cryostat sections of rabbit spleens by the twostep immunoperoxidase method described below. Clones producing antibodies reactive with splenic macrophages were selected, cloned twice by the limiting dilution procedure, and processed for mass culture. Ascites was obtained by injection of 5 x 106 hybrid cells into pristane-primed BALB/c mice. Determination of isotype The isotype of RbM2 was determined by the Ouchterlony method using a monoclonal typing kit (Miles, Naperville, IL).
Immunohistochemistry After killing with pentobarbital anaesthesia, the following tissues were excised from the rabbits and fixed with 2% periodate-lysine-paraformaldehyde (McLean & Nakane, 1974) for 4 hr: spleen, lymph nodes, thymus, liver, lungs, heart, aorta, stomach, Peyer's patches, kidneys, testes, ovaries, uterus, skeletal muscles, salivary glands, skin and brain. After washing in phosphate-buffered saline (PBS) containing a graded series of sucrose (10%, 15%, 20%), the tissues were rinsed for 1 hr in PBS containing 20% sucrose and 5% glycerol, then embedded in tissue embedding compound, and frozen in dry-ice acetone. Cryostat sections were cut at 6-8 gm thick and dried in air. The omentum was spread on albumin-coated slides without sectioning. Peritoneal macrophages and peripheral blood cells were prepared as cell smears and fixed in acetone. Immunostaining was performed as described previously (Takeya et al., 1987). In brief, the cryostat sections, spread omentum and cell smears were pretreated with 5 mm periodate for 10 min, washed with PBS, and immersed in 3 mm sodium borohydride for 30 min, according to the method of Isobe et al. (1977), to inhibit endogenous peroxidase activity. The specimens were incubated with culture supernatant or ascites diluted 1: 1000 in PBS as primary antibody for 1 hr, washed well with PBS, and covered with species-specific sheep anti-mouse Ig [F(ab')2] conjugated with peroxidase (Amersham, Amersham, Bucks, U.K.) as secondary antibody for 1 hr. To visualize peroxidase activity, the specimens were incubated with 3,3'-diaminobenzidine (DAB) H202 as substrate. Counter staining was done with haematoxylin for tissue specimens and with the Giemsa method for smears. As the control, tissue specimens were incubated with non-immunized mouse serum or PBS instead of primary antibody, and then processed by the same procedure described above. Tissues of species other than mouse were processed and stained in the same manner as for rabbit tissues. In immunostaining mouse tissues, biotinylated primary antibodies were applied first, followed by treatment with avidin-biotin-peroxidase complex. Visualization of peroxidase activity was performed as mentioned above. Immunoelectron microscopy Rabbit peritoneal exudate cells obtained as described above were incubated on Petri dishes with RPMI-1640 supplemented with 10% fetal calf serum at 370 for 30 min. After removing the non-adherent cells, the adherent cells were fixed with 0 05% glutaraldehyde in 0-165 M cacodylate buffer, pH 7-4, for 10 min. Immunostaining was performed as described above, with minor
modification. Incubation with the mAb was performed overnight, and that with the second antibody was done for 3 hr. After detection of peroxidase activity with DAB, the cells were postfixed with 1% osmium tetroxide for 1 hr, dehydrated through a graded series of ethanols, and detached from the dishes by adding propylene oxide. The cell pellets were embedded in Epon 128 and sectioned by an Ultrotome Nova (LKB, Uppsala, Sweden). To collect peripheral blood monocytes, blood was obtained by cardiac puncture from the rabbits under pentobarbital anaesthesia and decoagulated with heparin. Collected blood was diluted two-fold with RPMI-1640. After gently adding Lymphoprep (Nycomed AS, Oslo, Norway), the diluted blood was centrifuged at 400 g for 30 min to separate the mononuclear cell layer. This cell layer was fixed, immunostained, and processed for electron microscopy in the same manner mentioned above. The sections were observed under an electron microscope H-300 (Hitachi, Tokyo) without poststaining. Enzyme cytochemistry For subcellular demonstration of acid phosphatase, adherent peritoneal macrophages obtained as described above were fixed in 2 5% glutaraldehyde for 1 hr and then subjected to the Gomori method as modified by Barka & Anderson (1963). Latex particle endocytosis Adherent peritoneal macrophages obtained as above were incubated for 30 min at 0°, 16° and 370 after the addition of Latex particles with different diameters (0-1 ,um and 0-8 ,um; Sigma) and submitted to immunoelectron microscopy as described above. To inhibit phagocytosis, adherent cells were incubated 30 min at 370 in the culture medium containing 20 ,ug/ ml of Cytochalasin B (Sigma) and processed for immunoelectron microscopy. Immune phagocytosis Adherent peritoneal macrophages were incubated for 1 hr at 370 or below 160 in the medium containing the same volume of sheep erythrocytes (E) coated with anti-E antibody (IgG) (IgG-EA) adjusted to 1 x 108/ml. They were then fixed as described above and subjected to immunoelectron microscopy. Estimation of molecular weight To estimate the molecular weight (MW) of RbM2-recognized antigen, proteins were extracted from rabbit peritoneal macrophages with 1% n-octyl-fl-glucoside in phosphate buffer, dissolved in sample buffer [0-5 M Tris-HCl buffer 2 5 ml, 10% sodium dodecyl sulphate (SDS) 4 ml, glycerin 2 ml, 2-mercaptoethanol 1 ml, 0 05% bromophenol blue 0 54 ml and distilled water 10 ml], and boiled for 4 min. Using SDS-PAGE mini (Tefco, Tokyo), electrophoresis was performed on an 8% SDSPAGE gel. Proteins were blotted for I hr at 40 on nitrocellulose membranes (Millipore, Bedford, MA). Membranes were immunostained using the blotting detection kit (Amersham). Briefly, after the membranes were blocked with 10% dried milk solution, they were incubated with primary antibody solution for I hr at room temperature, followed by incubations with a biotinylated anti-mouse immunoglobulin antibody for 20 min and then with a streptavidin-biotinylated alkaline phosphatase complex for 20 min. Alkaline phosphatase activity was visualized by incubating the membranes with substrate solution that
Monoclonal antibody RbM2 had been made by adding a drop of nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate solutions into each 10 ml of diethanolamine buffer. Whole protein patterns of rabbit peritoneal macrophages and a molecular weight standard were stained with Amido black. RESULTS Isotype of the mAb On the frozen sections, three out of 35 hybridomas were found to be producing antibodies reactive with splenic macrophages. In the present paper, one of them was chosen, designated RbM2, and further characterized. The isotype of RbM2 was IgG 1.
Distribution, morphology and immunohistochemical characterization of RbM2-reactive cells The mAb RbM2 was found to react exclusively with phagocytes in different organs and tissues, as determined by immunohistochemistry. The cells were defined as macrophages based on their morphology and localization in the various compartments of the lymphoid and non-lymphoid organs. In general, the larger and more phagocytic the macrophages became, the more intensely they stained with the antibody. At high magnification, intense reactivity was observed in the cytoplasmic granules of phagocytosing macrophages. In the red pulp of the spleen, RbM2 reacted intensely with cordal macrophages, which were large and round or polygonal (Fig. la). In the white pulp, a few polygonal macrophages were reactive in the germinal centre and mantle zone, but there was no staining of marginal metallophils, IDC in the periarteriolar lymphatic sheaths (PALS), or FDC in the germinal centre. In the lymph nodes, the reaction for RbM2 was limited to sinus macrophages alone, particularly those in the medullary sinuses, and to macrophages in the lymphoid follicles (Fig. Ib). In the lymph sinuses, RbM2-reactive macrophages were large and mostly polygonal. In the lymphoid follicles, tingible body macrophages also stained positively. However, no reactivity was detected in any dendritic cells, including IDC in the paracortical areas and FDC in the germinal centre. In the thymus, polygonal macrophages reactive with RbM2 distributed sporadically (Fig. lc), but thymic epithelia or dendritic cells showed no reactivity. In the Peyer's patches, intense reactivity was detected in round or polygonal macrophages in the lymphoid follicles, particularly in the germinal centre. Similar positive cells were found in the dome of lymphoid follicles and interfollicular areas (Fig. I d), but cells with dendritic morphology did not stain. In the liver, Kupffer cells reacted with RbM2, but not with the sinusoidal endothelia, perisinusoidal fat-storing cells or parenchymal cells (Fig. Ie). In the lungs, alveolar macrophages showed a positive reaction for the antibody (Fig. lf), and a few interstitial macrophages were also reactive with it. In the skin, polygonal or spindle dermal macrophages were stained positively, but no reactivity was detected in any epidermal dendritic cells, including Langerhan's cells. In the brain, a positive reaction for RbM2 was demonstrated in round or polygonal macrophages in the subarachnoid space and in some perivascular spindle cells in the perivascular space, but resting microglia were unreactive.
515
Peritoneal macrophages reacted with RbM2 (Fig. Ig, h) and intense reactivity was found in their cytoplasmic granules. However, their cytoplasm was often stained diffusely due to the diffusion of immunoreactive products for RbM2 from the cytoplasmic granules into the cytoplasm, which occurs in immunostaining, probably by visualization of peroxidase activity. In the omentum, RbM2-reactive macrophages mostly appeared round or polygonal (Fig. Ig), but a few were spindle. In the milky spots, a large number of variable-sized, round cells accumulated, mostly reacting with the antibody. Further, RbM2-positive macrophages were dispersed in the subcutaneous tissues, the mucosa of the gastrointestinal tract, the endometrium, and the interstitial tissues of different organs and tissues. Besides, monocytes stained positively with RbM2. Polymorphonuclear leucocytes showed no reactivity, and lymphocytes, erythrocytes or other kinds of cells, including epithelial, endothelial, neuronal and muscle cells, did not stain with the antibody. These results indicate that this mAb reacts with the monocyte/macrophage population alone and not with dendritic cells. Over 90% of the peritoneal macrophages in ascites and the monocytes in the peripheral blood also reacted with RbM2. However, it was not reactive with the macrophages of mice, rats, guinea-pigs, hamsters, goats or humans. Intracellular localization of reactivity with RbM2 in macrophages and monocytes Immunoelectron microscopy revealed that reaction products of RbM2 were confined to variable-sized vacuoles of peritoneal macrophages (Fig. 2a), which were consistent with the lightmicroscopic immunohistochemical findings mentioned above. RbM2-reactive vacuoles were mostly large and irregular, and some contained certain digested or degraded materials, suggesting secondary lysosomes. Although reactivity was also found around some large vacuoles, it was considered to be an artifact caused by diffusion of the reagent during immunostaining. Reactivity with the antibody was also detected in primary lysosomes, but pinocytic vesicles, tubular structures and endosomes (receptosomes), with or without multivesicular profiles, were not reactive with the mAb. In addition, the cell membrane, nuclear envelope and intracellular organelles other than lysosomes showed no reactivity. In monocytes, the localization pattern of RbM2 was essentially the same. Ultrastructural cytochemistry of acid phosphatase activity revealed a localization pattern similar to that of the reactivity with RbM2 (Fig. 2b). This result indicates that this mAb recognizes lysosomes and their related compartments. In vitro investigation of the reactivity with RbM2 in macrophages during endocytosis On incubation at 00 for 30 min in the medium containing Latex particles with diameters of 0-8 gm and 0l 1 m, no specific binding of these particles was found on the cell surface of peritoneal macrophages. On temperature shifting to 370, they started endocytosing Latex particles in the fluid phase. At 5 min after temperature shifting, the macrophages ingested many Latex particles, around most of which the reaction products of RbM2 were demonstrated immunoelectron microscopically (Fig. 3a). However, when incubated in the same medium below
516
r_E1 .^_:.
Y. Shimokawa et al.
g
.I
._
_
_
:l.5+:** :.
*4
_ t
*
*:_
|
.uS
::n..
:^s
sw
w
,'!:
t
I_
._a
E
t
t
,,,_
.. ^
^:: _
..
#
.:
_
,_
w
Figure 1. Distribution of RbM2-positive cells in various organs and tissues of normal rabbits. (a) Macrophages in the red pulp (rp) and white pulp (wp) of the spleen ( x 180). (b) Macrophages in the medullary sinuses (ms), and tingible body macrophages in the lymph follicle (fo) of the lymph node ( x 160). (c) Macrophages in the thymic cortex and medulla ( x 110). (d) Macrophages in the lymph follicles and interfollicular regions of the Peyer's patch (x 70). (e) Kupffer cells in the hepatic sinusoids (x 180). (f) Alveolar macrophages (x 400). (g) Macrophages in the milky spot (ms) of the omentum (x 110). (h) Peritoneal macrophages (arrow); lymphocytes and granulocytes are negative (arrowhead) (x 540). Staining with two-step immunoperoxidase method using RbM2.
517
Monoclonal antibody RbM2 DISCUSSION
Figure 2. (a) Peritoneal macrophages exhibit a positive reaction with RbM2 in phagocytic vacuoles and primary lysosomes (arrow). Immunoelectron microscopy with RbM2 (bar= 1 ,m). (b) In the macrophages, phagocytic vacuoles (heterolysosomes) and primary lysosomes (arrow) are positive for acid phosphatase. Barka & Anderson (1963) method (bar= 1 ,m).
16", the temperature known to inhibit lysosomal fusion, the ingestion of Latex particles by the cells decreased, and no reactivity with the mAb was detected in any vesicles or vacuoles containing the particles (Fig. 3b). These results indicate that among Latex particles internalized by the macrophages via fluid phase endocytosis, RbM2 recognizes only those fused with lysosomes. When cultured in the medium to which Cytochalasin B was added to inhibit phagocytosis, the macrophages ingested only the Latex particles with a diameter of 0 1 ,um, not those of 0-8 ,m. With time, the Latex particles were pinocytosed as a single particle, aggregated and fused with each other into large phagocytic vacuoles. RbM2 reacted with such large vacuoles but not with the pinocytic vesicles containing a single particle (Fig. 3c). Cytochemically, acid phosphatase activity was demonstrated in these large phagocytic vacuoles alone. This indicates that they are heterolysosomes. In the experiment of immune phagocytosis with IgG-EA, reactivity with RbM2 was detected around sheep erythrocytes phagocytosed by macrophages (Fig. 3d). MW of RbM2-recognized antigen
RbM2 immunostained a protein band of 50,000 MW in comparison with the MW standard stained with Amido black (Fig. 4).
The mAb RbM2 reacts with rabbit macrophages, as determined by tissue distribution and cell morphology. It is also reactive with peripheral blood monocytes, but not with granulocytes or other blood cells. Dendritic accessory cells of the T- or B-cell immune response, such as IDC in the lymphoid T zone, epidermal Langerhan's cells, or FDC within the lymph follicles, show no reactivity with RbM2. RbM2 showed no cross-reaction to macrophages in lymph nodes of humans and animals other than rabbits. Thus, RbM2 is a suitable reagent to differentiate the monocyte/macrophage population from the dendritic accessory cell populations in rabbits. In previous studies, two anti-rabbit macrophage mAb were produced, which were termed SRM 1 and RAM 11 (Watanabe et al., 1985; Tsukada et al., 1986). Compared to RbM2, they show a nearly identical reactivity with macrophages in distribution, but they differ from each other in isotype, recognition site and kind of immunoreactive cells other than macrophages. Our approach by immunoelectron microscopy demonstrated that RbM2 is confined to phagocytic vacuoles or to cytoplasmic granules in the monocyte/macrophage population. Ultrastructural cytochemistry for the demonstration of acid phosphatase revealed that RbM2-positive phagocytic vacuoles or cytoplasmic granules are positive for acid phosphatase, thus indicating that they are consistent with secondary or primary lysosomes. Near the cell membrane, we found some vacuoles or vesicles unreactive with RbM2 and showing no positive reaction for acid phosphatase activity. These vacuoles or vesicles are considered to be endocytic vesicles or vacuoles prior to fusion with lysosomes. To demonstrate the recognition site of RbM2 more clearly, we performed various experiments of endocytosis by rabbit peritoneal macrophages and monocytes. Endocytosis is discriminated into phagocytosis and pinocytosis depending on the size of the particles ingested, and phagocytosis is defined as the internalization of large particles greater than 0 1 ,um, a process known to be inhibited by Cytochalasin B (Wills et al., 1972). Based on these facts, we examined the immunoreactivity of RbM2 in the macrophages and monocytes during endocytosis of particles of different sizes. As a result, immunoreactivity with RbM2 in the cells was confirmed to be independent of the type of endocytosis, i.e. phagocytosis or pinocytosis and fluid phase endocytosis or receptor-mediated endocytosis. This was because almost all the heterolysosomes were labelled by RbM2 in the processes of ingestion of variously sized Latex particles and IgG-sensitized erythrocytes. In all these endocytosis experiments, there was no immunoreactivity with RbM2 around the particles at temperatures below 16°, but on temperature shift to 370 there was. These data indicate that RbM2 recognizes lysosomes and not endosomes or endocytic vesicles. In secondary lysosomes, immunoreactivity with RbM2 was observed more intensely near the lysosomal membrane, suggesting that this mAb recognizes a lysosomal membrane antigen. From the fact that the mAb does not react with any other kind ofcell, even lysosome-rich cells such as hepatocytes, neuronal cells, or renal tubular epithelia, it is suggested that this antigen is a glycoprotein of 50,000 MW specifically localized on the lysosomal membrane of the monocyte/macrophage population alone. Furthermore, we speculate that the antigen is one of the
Y. Shimokawa et al.
518
aJ4!~~~~~ Figure 3. Immunoreactivity of peritoneal macrophages with RbM2 during endocytosis. (a) On incubation with Latex particles (0 8 ium) for 20 mmn at 370, some particle-containing vacuoles react with RbM2 (arrow), and others are unreactive (arrowhead) (bar = 1 jim). (b) On incubation below 16°, almost all the Latex particles show no reaction against RbM2 (bar = 1 tim). (c) On incubation at 370 after adding Latex particles (0-1 jim) to the medium, large phagocytic vacuoles containing many particles are reactive with RbM2, and single particle-containing endosomes are unreactive (bar = 1 jim) (arrows). (d) Incubation with sensitized sheep erythrocytes at 370 for 20 min. Two erythrocytes are ingested by the macrophage: one is a heterolysosome reacting with RbM2, the other is unreactive during the endosomal stage (arrow) (bar = 1 jim). Immunoelectron microscopy with RbM2.
(a)
50,000
(b) (c) .MW _ 1111
E
I97400 -|966,200 42eOO
I 421,500 4
14,400
Figure 4. Molecular weight of an antigen recognized by RbM2. (a) RbM2 immunostaining. (b) Whole protein pattern of peritoneal macrophages. (c) Molecular weight standard.
essential components of the lysosomal membrane and is increased in secondary lysosomes, because RbM2 is expressed in not only secondary lysosomes but also primary lysosomes, more prominently in heterolysosomes (secondary lysosomes), and irrespective of type of endocytosis. To elucidate the property of the lysosomal membrane antigen, biochemical and immunological investigations are required.
ACKNOWLEDGMENTS This work was done at the Second Department of Pathology, Kumamoto University Medical School, and supported in part by a grant-inaid for scientific research from the Ministry of Education, Welfare and
Culture, Japan.
REFERENCES ANDERSON C.L., GuYRE P.M., WHITIN J.C., RYAN D.H., LOONEY R.J. & FANGER M.W. (1986) Monoclonal antibodies to Fc receptors for IgG on human mononuclear phagocytes: antibody characterization and induction of superoxide production in a monocyte cell line. J. biol.
Chem. 261, 12856. AUSTYN J.M. & GORDON S. (1981) F4/80, a monoclonal antibody directed specifically against the mouse macrophage. Eur. J. Immunol. 11, 805. BARKA T. & ANDERSON P.J. (1963) Histochemistry: Theory, Practice, and Bibliography, p. 239. Harper & Row, New York. CHEN J.W., MURPHY T.L., WILLINGHAM M.C., PASTAN I. & AUGUST J.T. (1985) Identification of two lysosomal membrane glycoproteins. J. Cell Biol. 101, 85. FLoTTE T.J., SPRINGER T.A. & THORBECKE G.J. (1983) Dendritic cell and macrophage staining by monoclonal antibodies in tissue sections and epidermal sheets. Am. J. Pathol. 111, 112. ISOBE Y., CHEN S.T., NAKANE P.K. & BROWN W.R. (1977) Studies on translocation of immunoglobulins across intestinal epithelium. I. Improvements in the peroxidase-labelled antibody method for application to study of human intestinal mucosa. Acta Histochem. Cytochem. 10, 161. KOHLER G. & MILSTEIN C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature (Lond.), 256,495. KRAAL G., SHIAMATEY-KOOLMA R., HOFFER M., BARKER D. & SCHEPER R. (1988) Histochemical identification of guinea-pig macrophages by monoclonal antibody MR-1. Immunology, 65, 523. KREIPE H., RADZUN H.J., PARWARESCH M.R., HAISLIP A. & HANSMANN M.L. (1987) Ki-M7 monoclonal antibody specific for myelomonocytic cell lineage and macrophages in human. J. Histochem. Cytochem. 35, 1117.
Monoclonal antibody RbM2 MCLEAN I.W. & NAKANE P.K. (1974) Periodate-lysine-paraformaldehyde fixative. A new fixative for immunoelectron microscopy. J. Histochem. Cytochem. 22, 1077. MANE S.M., MARZELLA L., BAINTON D.F., HOLT V.K., CHA Y.,
HILDRETH J.K. & AUGUST J.T. (1989) Purification and characterization of human lysosomal membrane glycoproteins. Arch. Biochem. Biophys. 268, 360. PARWARESCH M.R., RADZUN H.J., HANSMANN M.L. & PETERS K.P.
(1983) Monoclonal antibody Ki-M4 specifically recognizes human dendritic reticulum cells (follicular dendritic cells) and their possible precursor in blood. Blood, 62, 585. PARWARESCH M.R., RADZUN H.J., KREIPE H., HANSMANN M.L. &
BARTH J. (1986) Monocyte/macrophage-reactive monoclonal antibody Ki-M6 recognizes an intracytoplasmic antigen. Am. J. Pathol. 124, 141. RADZUN H.J., KREIPE H., BODEWADT S., HANSMANN M.L., BARTH J. &
PARWARESCH M.R. (1987) Ki-M8 monoclonal antibody reactive with an intracytoplasmic antigen of monocyte/macrophage lineage. Blood, 69, 1320. TAKEYA M., HSIAo L., SHIMOKAWA Y. & TAKAHASHI K. (1989) Heterogeneity of rat macrophages recognized by monoclonal anti-
bodies: An immunohistochemical and immunoelectron microscopic study. J. Histochem. Cytochem. 37, 635.
519
TAKEYA M., HSIAo L. & TAKAHASHI K. (1987) A new monoclonal antibody, TRPM-3, binds specifically to certain rat macrophage populations: Immunohistochemical and immunoelectron microscopic analysis. J. Leukocyte Biol. 41, 187. TSUKADA T., ROSENFELD M., Ross R. & GOWN A.M. (1986) Immunocytochemical analysis of cellular components in atherosclerotic lesions. Use of monoclonal antibodies with the Watanabe and fat-fed rabbit. Arteriosclerosis, 6, 601. UKAi K., TERASHIMA K., Fujii Y. & IMAI Y. (1988) A new monoclonal antibody, UFT-4, reacting with rat Kupffer cells. Immunohistochemical and immunoelectron microscopical analysis with reestimation of the reticuloendothelial system. Acta Pathol. Jpn. 38, 1391. WATANABE T., HIRATA M., YOSHIKAWA Y., NAGAFUCHI Y., ToYOSHIMA H. & WATANABE T. (1985) Role of macrophages in atherosclerosis. Sequential observations ofcholesterol-induced rabbit aortic lesion by the immunoperoxidase technique using monoclonal antimacrophage antibody. Lab. Invest. 53, 80. WILLS E.J., DAVIES P., ALLISON A.C. & HASWELL A.D. (1972) Cytochalasin B fails to inhibit pinocytosis by macrophages. Nature New Biol. 240, 58.
