Immunology 1990 70 262-271

A novel monoclonal antibody, Mar 1, directed specifically against mononuclear phagocyte system cells in rats A. YAMASHITA, Y. HATTORI, M. KOTANI,* M. MIYASAKA* & T. FUKUMOTOt Department of Anatomy, Hamamatsu University School of Medicine, Hamamatsu, Japan

Acceptedfor publication 14 February 1990

SUMMARY Three different monoclonal antibodies (mAb), designated Mar 1, Mar 2, and Mar 3, recognizing three distinct novel antigen molecules expressed preferentially in rat macrophages, were produced by the hybridoma technique. Binding of these mAb to isolated cells or fixed cells was detected by radioactive binding assay, immunohistochemical technique and flow cytometry. Mar 1 binds specifically to the cells constituting the mononuclear phagocyte system (MPS), but not to granulocytes nor endocytosis-positive cells from non-lymphoid tissues. Mar 2 and Mar 3 recognize both the former and the latter. The isotypes of Mar 1, Mar 2 and Mar 3 were defined as IgGl, IgGl and IgG2b, respectively. These mAb were species specific, allo-non-specific and not cytotoxic for rat peritoneal macrophages. Immunoelectron microscopic observation demonstrated that Mar 1-3 antigens are located on both surface membrane and cytoplasmic membrane structures of peritoneal macrophages, particularly on the limiting membrane of phagocytic small vesicles and large phagosomes. Immunoprecipitation experiments demonstrated that the apparent molecular weights (MW) of the reactive antigens of Mar 1, Mar 2 and Mar 3 are 95,000, 100,000 and 55,000 and 27,000, respectively. These findings indicate that all of Mar 1-3 mAb have considerable value in the identification of rat phagocytes and that, of the three kinds of antigens detected with Mar 1-3, Mar I antigen is a specific marker for identification of the cells constituting the MPS and may offer the means to assess the functional capability and differentiation process of the macrophage populations.

1981; Robinson, White & Mason, 1986). Most of these mAb are directed against both monocytic cells and granulocytic cells. However, there are a few mAb that preferentially recognize common antigenic determinants of only macrophage populations, and thus overcome the problem of intra-populational

INTRODUCTION It is well known that macrophage populations show a distinct intra-populational heterogeneity with regard to morphology, stage of differentiation, enzyme activity, cell-surface property, stage of activity, function and many other features (Debakker & Daems, 1981; Marahan, 1980). A large number of monoclonal antibodies (mAb) specifically recognizing the phenotypic appearance of heterogeneous macrophage populations has been produced in human (Todd & Schlossman, 1982; Wright et al., 1983) and rodents (Springer et al., 1979; Austyn & Gordon,

heterogeneity. Recently, three different mAb, designated Mar 1, Mar 2 and Mar 3, were produced recognizing rat macrophage populations. The present study reports that Mar 1 binds specifically to rat mononuclear phagocyte populations but not to granulocytic cells, and thus provides a highly specific novel marker for monocytes and macrophages in rats. Furthermore, the study demonstrates that Mar 2 and Mar 3 recognize both cell populations belonging to the mononuclear phagocyte system (MPS) (Van Furth et al., 1972) and some populations of endocytosis-positive cells in non-lymphoid tissues. Their characterization suggests their potential utility in defining both the structure of common antigen molecules in mononuclear phagocyte populations and the process of macrophage differentiation.

* Present address: Dept. of Immunology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan. t Present address: Dept. of Anatomy, Yamaguchi University School of Medicine, Ube, Japan. Abbreviations: BSA, bovine serum albumin; DAB, diaminobenzidine tetrahydrochloride; i.p., intraperitoneally; mAb, monoclonal antibody(ies); MPS, mononuclear phagocyte system; MW, molecular weight; PBS, phosphate-buffered saline; PCV, post-capillary venules; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; TBS, Tris-HCl balanced saline. Correspondence: Dr A. Yamashita, Dept. of Anatomy, Hamamatsu University School of Medicine, Handa-ChM 3600, Hamamatsu-Shi,

MATERIALS AND METHODS Animals Male BALB/c (H-2b) mice, Lewis (RT1') rats and Wistar-SLC (RTlk) rats were obtained from Shizud6ky6 Ltd (Shizuoka).

Shizuoka-Ken, 431-31, Japan.

262

Mononuclear phagocyte system Inbred male DA (RTla) and HO(RT1C) rats were obtained from the John Curtin School of Medical Research, Canberra, Australia. They were kept under controlled environmental conditions and used when 7-10 weeks of age. Preparation of mAb The cloned hybrid cell lines secreting Mar 1, Mar 2 and Mar 3 mAb were derived from fusions between mouse spleen cells and the mouse myeloma P3-NS l/I-Ag-l(kindly provided by Dr C. Milstein, Cambridge, UK.). BALB/c mice were primed intraperitoneally (i.p.) with rat peritoneal macrophages (20 x 106) and boosted i.p. three times at weekly intervals. The spleens were harvested 5 days after the last injection. Fusions were done following the method of Kohler & Milstein (1975), modified after McMaster & Williams (1979), except that the cells were cloned and recloned by adding 5 cells/ml to a rat thymocyte suspension containing 106 cells/ml and seeding 0-2 ml volumes/ well into microtitre plates. Antibody-secreting hybrids were detected by a radioactive binding assay, and screened on an EPICS-V flow cytometer (Coulter Ltd, Hialeah, FL) and by an indirect immunoperoxidase method. Cells reacting with putative macrophage that were detected by the above procedures were selected and cloned. Ascites fluid was obtained by i.p. injection of 5 x 106 hybrid cells into BALB/c mice. The isotype of the mAb was determine by Ouchterlony immunodiffusion in 1% agar using supernatants from cloned hybridoma cultures and rabbit antisera specific for mouse immunoglobulin isotypes. IgG was purified from ascites fluid from mice growing the appropriate hybridoma.

Cell preparation Rat peritoneal macrophages were obtained as previously described (Yamashita et al., 1978). Briefly, peritoneal exudate cells were collected from the peritoneal cavities of various strains of rats that had been given an i.p. injection of Streptococcus pyogenes preparation (OK 432; Chiugai Pharmacol. Co. Ltd, Tokyo; 0.01 mg dry weight) 4 days previously. Separation of macrophages from peritoneal exudate cells was performed by the glass adhesive technique. Peritoneal exudate cells were suspended in Hanks' solution, washed and resuspended in appropriate volumes of the medium. After 2 hr of incubation at 370, the non-adherent cells were separated from glass-adherent cells by extensive washing with the medium. Adherent cells were collected with a rubber policeman and adjusted to an appropriate concentration in Hanks' medium. The mean percentage of viability of resident macrophages and OK 432-activated macrophages was 88% and 92%, respectively, when tested by the trypan blue exclusion technique. Resident peritoneal macrophages were obtained without i.p. injection of OK 432, and purified as mentioned above. Granulocyte-enriched populations were harvested from the peritoneal exudate cells 4-6 hr after an i.p. injection of OK432. White blood cells were prepared from blood after bleeding rats out through the abdominal aorta. The heparinized aortal blood was diluted in an equal volume of 0-15 M NaCl and layered on top of I ml of Ficoll-Paque (Pharmacia Fine Chemicals Ltd, Tokyo). Following centrifugation at 200 g for 25 min, at room temperature, a lymphocye-rich layer at the interface and a granulocyte-rich layer at the bottom were transferred to a conical tube, and then contaminated red blood cells were removed from these fractions by lysing in Trisbuffered 0-83% (w/v) ammonium chloride. Bone marrow cells

263

were obtained by flushing the marrow cavity of femurs with Hanks' medium. Alveolar macrophages were obtained by the bronchial lavage method (Stewart, 1986). Cell suspensions obtained from spleens were enriched for dendritic cells according to the method of Steinman et al. (1979). Briefly, after treatment with collagenase, 0-5 mg/ml for 30 min at 37°, the spleens were teased with pincettes and incubated for 2 hr in Hanks' medium containing 2% fetal calf serum, and then the non-adherent cells were dislodged with gentle pasteur pipetting. To purify the dendritic cell fraction, the adherent monolayers were cultured overnight in 5% fetal calf serum-Hanks' medium. Most of the dendritic cells were dislodged into a suspension of viable cells using a gentle pipetting procedure. The enrichment procedure of dendritic cells was monitored by phase contrast microscope and light microscope (dendritic cells are irregular in shape, extending slender processes in many directions and without surface ruffles) and also by positive expression of Ia antigen on the cell surface, detected by indirect immunoperoxidase staining using anti-Ia mAb (MRC OX6; Sera Lab. Ltd, Crawley Down, Sussex, U.K.).

Binding assay Indirect radioimmunoassays under saturating conditions were performed according to the method of Mason & Williams (1980) to detect the hybridoma clone secreting mAb molecules that bound to rat macrophages. Briefly, tissue culture supernatant was incubated with an equal volume of freshly prepared OK 432-activated macrophages which were obtained from various strains of rats. The cell-bound mAb were detected with '25I-rabbit F(ab')2 anti-mouse IgG using a Hitachi automatic well counter. The rabbit F(ab')2 anti-mouse IgG (Sera Lab. Ltd) was previously labelled with 1251 by the chloramine T method (Mason & Williams, 1980).

Immunofluorescence staining Indirect immunofluorescence staining was carried out according to the method of Barclay (1981). The stained cells were analysed on a EPICS-V flow cytometer. Immunocytohistochemical staining Cytocentrifuge preparations made by slow centrifugation (Cytospin 2; Shandon, Southern Products, Cheshire, U.K.) or cryostat sections of various organs were air-dried and fixed in acetone for 10 min. The slides were incubated with the mouse anti-rat macrophages mAb Mar 1-3 for 1 hr after washing them in 0-01 M PBS (pH 7-4). In these instances, the negative controls were P3X supernatant (IgGI isotype) for Mar 1-2 mAb and anti-sheep T-cell mAb 197 (IgG2b) for Mar 3, respectively (both kindly provided by Dr W. R. Hein, Basel, Switzerland). The cells binding the mAb were detected by an indirect immunoperoxidase method (Barclay, 1981) or an indirect immunoalkaline phosphatase method (Moir et al., 1983) after further washing in PBS.

Immunoperoxidase staining Slides were covered with peroxidase-conjugated rabbit F (ab')2 anti-mouse IgG (Cappel Labs Inc., PA) diluted 1: 100 in 2% normal rat serum for I hr. After washing in PBS, the slides were stained for peroxidase activity with 3,3'-diaminobenzidinetetrahydrochloride (DAB, 0-5 mg/ml Tris-HCl buffer, pH 7 6, containing 0001% H202); Sigma Chemical Co., St Louis, MO

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and 0 01 M imidazole (Sigma Chemical Co.) in 0 01 M Tris-HCl balanced saline (TBS) solution. Immunoalkaline phosphatase staining Slides were incubated with the alkaline phosphatase-conjugated rabbit F(ab')2 anti-mouse IgG(Zymed Labs Inc., South San Francisco, CA) for 30 min. After washing in cold PBS, the slides were incubated again with the alkaline phosphatase-conjugated goat F(ab')2 anti-rabbit IgG (Zymed Co.). The alkaline phosphatase conjugate was detected by the Fast red technique (Moir et al., 1983). When necessary, endogenous alkaline phosphatase activity was eliminated with a solution of I mm levamisole substrate. The slides were lightly counterstained with Harris' haematoxylin. Immunoelectron microscopic assay Pellets of peritoneal exudate macrophages were fixed with 0-1% glutaraldehyde-5% paraformaldehyde-0 1 M phosphate buffer (pH 7 4) for 10 min and washed first with 0 1 M glycine in PBS for 30 min and then with 0-05% Tween 20-0-25% bovine serum albumin (BSA)-00 I M TBS solution. Fixed cells were incubated with mAb at 40 for I hr. In this instance, P3X supernatant and 197 mAb were used also at the negative controls. After washing in 0-05% Tween 20-0-25% BSA-0-01 M TBS, cells were covered with a 1: 200 dilution of peroxidase-conjugated rabbit serum for I hr. They were washed in PBS, fixed in 2% glutaraldehyde-PBS for 20 min, and then reacted with DAB containing 0-01 % H202. Ultrathin sections were cut with an LKB ultramicrotome after the cells were post-fixed with OS04 and embedded in Epon.

Immunoprecipitation and SDS-PAGE Peritoneal exudate macrophages were lactoperoxidase radioiodinated according to the method of Cone & Marchalonis (1974). Radiolabelled macrophages were treated with TBScontaining 1% NP40 detergent at 40 for 20 min and dissolved in 0 5 ml of lysis buffer (100 mm NaCl, 0 I % sodium deoxycholate, 0 1% SDS, 1% NP40) at 4° overnight. Following centrifugation, antigen-antibody complexes were precipitated with the culture supernatant of a hybridoma clone and goat anti-mouse IgG, according to the technique of Tamura et al. (1984). Immunoprecipitated molecules were released by boiling for 3 min at 1000 with 5% 2-mercaptoethanol (reduced) or without 2-mercaptoethanol (unreduced), and applied to 7 5-15% linear gradient SDS-PAGE gels (Laemmli, 1970). Following electrophoresis, the slab gels were dried and exposed at - 70° for 10 days, and then autoradiographed. The molecular weight (MW) markers (Sigma Chemical Co.) used were: myosin 200,000 MW, phosphorylase B 92,000 MW, BSA 69,000 MW, ovalbumin 46,000 MW, carbonic anhydrase 30,000 MW and lysozyme 14,3000 MW.

RESULTS

Specific binding of Mar series mAb to peritoneal macrophages Hybridoma clones secreting antibodies that bound only to macrophages were detected by an indirect radioimmunoassay and screened on an EPICS-flow cytometer and by immunoperoxidase staining. Three clones reacting with peritoneal macrophages. mAb Mar 3 bound to macrophages but also weakly to neutrophils.

respectively, were found to be remarkably restricted in specificity. mAb Mar 1 and Mar 2 bound only to rat peritoneal macrophages. mab Mar 3 bound to macrophages but also weakly to neutrophils. Peritioneal macrophages from DA rats and other inbred rat strains, HO, Lewis or Wistar-Slc, were tested with the mAb Mar series by indirect radioimmunoassay. No significant differences were found in the extent to which the mAb bound to cells from these different strains. No differences were found in the binding of the mAb to cells from male or female rats from the four strains. No peritoneal macrophages obtained from guinea-pigs and mice, and no blood monocytes or bronchial lavage macrophages from humans, bound any of the Mar series of mAb. Thus, these data indicate that each of the mAb from the Mar series defines a determinant which is restricted to macrophages from the rat. This is not a polymorphic determinant. The mAb from the Mar series are thus species specfic but show no strain or sex specificity within the species. Figure 1 shows the representative fluorescence profiles of 4-day OX 432-activated peritoneal cells labelled with Mar I or Mar 2 and analysed by EPICS-V. Each mAb labelled over 99% of adherent macrophages and approximately 90% of 'gated' macrophages, compared with 3% of the control. No binding of either mAb to lymphocytes was detected by EPICS. Almost similar EPICS profiles were observed when peritoneal macrophages were labelled with Mar 3, whether they had been activated with stimulants or not. These results obtained by EPICS analysis were highly reproducible and indicate that Mar 1, Mar 2 Mar 3 specifically label both resident and activated rat macrophages. The fact that the fluorescence intensity and percentage labelling of 'gated' macrophages labelled with the Mar series mAb, as shown in Fig. 1, were lower than that of adherent macrophages suggests that the degree of antigen expression on the macrophage-surface membrane may be dependent on the conditions of the cells harvested. A probable explanation for the lower intensity is that the adhesion of macrophages incubated for 2 hr at 370 before labelling results in increased surface expression of the antigen, either from de novo synthesis or by transport from the intracellular pools, since antigenic modulation has been produced in vitro by inhibiting macrophage adherence during incubation with anti-mouse macrophage mAb 3A33 (Collet et al., 1985). Further study would be required to clarify the mechanism of antigenic modulation of macrophage-surface antigen by the Mar series mAb. The isotypes of mAb Mar 1, Mar 2 and Mar 3 were defined as IgGl, IgGl and IgG2b, respectively. These mAb were not directly cytotoxic for rat peritoneal macrophages in the presence of complement from rat, guinea-pig or mouse serum. Molecular nature of the antigens recognized by the Mar series mAb In order to determine the relative MW of the antigens recognized by mAb of the Mar series, cell-surface proteins on 4-day OK 432-activated peritoneal macrophages were radiolabelled using 1251. The radioactive molecules were then immunoprecipitated, analysed with SDS-PAGE and autoradiographed (Fig. 2). Mar 1 precipitated an antigen of 95,000 MW under non-reducing conditions. In the reduced state, the immunoprecipitates migrated as two definite bands of 95,000 MW and

Mononuclear phagocyte system (A)

(B) (a )

9966%

(b)

:; (b )

87.70%

(c) : : -.,

C-4.

'-.t'.~ 89.40/

(a )

265

99-8%

:~~~20/

;,.

Fluorescence intensity PFigure 1. Fluorescence profiles of peritoneal macrophages labelled with Mar 1 (A) and Mar 2 (B) and analysed using EPICS-V. 104 cells were analysed for their fluorescence intensity. (a) Profile of 4-day OK 432-activated macrophages enriched with the glass adhesive technique. (b) Profile of gated macrophages from exudate peritoneal cells, excluding lymphocytes, granulocytes and dead cells by the choice of suitable EPICS scatter gates, whose position on the oscilloscope is previously determined by each cell's light scattering (representative of cell size) and fluoresence intensity. (c) Profile of 'gated' macrophages from peritoneal exudate cells exposed to second antibody alone. The numbers given on each profile indicate the percentage of cells that fell into fluorescence channels above the broken vertical line.

55,000 MW. The two bands obtained after reduction of Mar 1 antigen did not seem to be due to technical problems, because rat IgG run on the same gels gave rise to the ordinary bands after the reducing condition used. On the other hand, Mar 2 precipitated antigen of 100,000 MW. Two polypeptides of 55,000 MW and 27,000 MW were seen in Mar 3 antigens. A heavier band at 55,000 MW seems to be identical to the lower MW band of Mar 1 under reduced conditions. The mobility of Mar 2 and Mar 3 antigens did not change significantly with reduction.

The types of cells bearing antigens recognized by Mar series mAb The data obtained by the immunocytochemical staining of cell smears and cytocentrifuge preparations are summarized in Table 1. Mar I strongly labelled all peritoneal macrophages from normal rats, peritoneal exudate macrophages from rats given an i.p. injection of OK 432 (Fig. 3a) and alveolar macrophages obtained by bronchial lavage. In addition, Mar 1 moderately labelled blood monocytes and macrophages associated with erythrocytic clusters in the bone marrow. It did not label erythrocytes, granulocytes, lymphocytes or platelets in the peripheral blood or in the bone marrow. The pattern of labelling seen with Mar 2 was almost identical to that of Mar 1. Mar 3 labelling was different from that of the two former mAb. This mAb was less specific for macrophages, and weakly labelled neutrophils from the blood, peritoneal cavity and bone marrow. Splenic dendritic cells showing their typical characteristics were stained very weakly by each of the Mar series mAb;

these antigens were detected in patches on the surface membrane and in the cytoplasm.

The distribution of Mar series antigens in tissues The data obtained by the immunohistochemical staining of cryostat tissue sections are summarized in Table 2. The labelling pattern seen with Mar 1 in the sections of lymphoid and nonlymphoid tissues was similar to that seen with Mar 3. In the thymus, large cells with strong acid phosphatase activity distributed throughout the cortex and medulla were stained with Mar 1; the density of positive-cell distribution was higher in the medulla than that in the cortex. And a rim of strong positive cells running in a line was found in the cortico-medullary junctions. It did not label Hassall's corpuscle epithelium of reticular cells. Labelling by Mar 3 was similar to Mar 1, except that this mAb weakly labelled Hassall's corpuscle epithelium. Tingible-body macrophages in the follicular areas of the lymph nodes and the spleen were labelled strongly with Mar 1 and Mar 3. Figure 3b shows a representative picture of tingible-body macrophages labelled strongly with Mar 1 which were distributed in the germinal centre of lymph follicles from the spleen. Free macrophages distributed in the cortex and medulla of lymph nodes and in the white pulp and red pulp of spleen were labelled with all three mAb. The location of the Mar series antigens on the lymphoid dendritic cells characterized by Steinman et al. (1979) was difficult to evaluate in tissue section because of the very weak staining of dendritic cells and the strong positivity of macrophages, although the positive staining in cell suspensions of spleen was defined by these mAb.

A. Yamashita et al.

266 (b)

(a)

(c)

(d) I

1

NR

R

NR

R

NPR

R

MW

200,000-

92,000 69,000 -

46,000-

30,000-

14,000Figure 2. Analysis by immunoprecipitation of the antigens recognized by Mar series mAb. Four-day OK 432-activated peritoneal cells (macrophage >90%) were radioiodinated with Na'25 I by the lactoperoxidase method, treated with detergent and immunoprecipitated with Mar I (b), Mar 2 (c), Mar 3 (d), positive control OX17 anti-Ia antien mAb (e) or negative control P3X supernatant (f). Materials obtained from the lysates by boiling were run, 5% 2-mercaptoethanol reduced (R) or non-reduced (NR), on 7 5%SDS-PAGE and detected by autoradiography. MW markers are indicated in (a).

Although endothelial cells lining the post-capillary venules (PCV) and lymph sinuses of lymph nodes were not reactive with Mar 1, Mar 3 weakly labelled the endothelium of the PCV. In the Peyer's patches, macrophages in both the follicular and interfollicular areas stained positive with both Mar 1 and Mar 3 mAb. The apical part of the dome epithelium and the absorptive epithelium in the bottom of the intestinal gland were weakly labelled with Mar 3, but not with Mar 1. In the liver, all Kupffer cells were stained with both mAb but the hepatocytes were not. Mesangial cells in the renal glomeruli were stained very weakly with only Mar 1. In contrast, Mar 3 labelled the apical surface of the proximal tubules. Alveolar macrophages and interstitial macrophages in the lung were labelled strongly by both Mar 1 and Mar 2 mAb. Langerhans' cells in the epidermis were negative to all three mAb, although most of the interstitial macrophages in the dermis were strongly positive. The follicular epithelium on the apical surface of the thyroid gland was labelled with Mar 3 and Mar 2, but not with Mar 1. In the brain, the granular perithelium covering the endothelium of small blood vessels, particularly in the cortical areas (Mato et al., 1981), was stained strongly with both Mar 1 and Mar 3 mAb and weakly with Mar 2. Most of microglia were positive with Mar 3, but not with Mar 1 nor Mar 2. Hofbauer cells in the placenta were positive with all three mAb. Mar 3 labelled the apical surface of the epithelium of the choroid plexus in the brain, but Mar 1 did not. The pattern of labelling with Mar 2 in the tissues tested was different from the labelling pattern seen with Mar 1 and Mar 3, even though the labelling pattern of the various free macrophage populations was very similar for all three mAb (Table 1). The tingible body macrophages in the follicles of the lymph

nodes, spleens and Peyer's patches were very weakly labelled with Mar 2, although the two other mAb labelled these cells strongly (Table 2). And Mar 2 stained only a few cells very weakly throughout the thymus. As with Mar 1, Mar 2 did not show definite labelling of the tracheal epithelium, the endothelium of the PCV, the dome epithelium of the Peyer's patches or the tubular epithelium of the kidney. As with Mar 3, Mar 2 showed definite labelling of the follicular epithelium of the thyroid gland and the apical surface of epithelium of the choroid plexus (Table 2). When control sections were cultured with P3X supernatant or 197 mAb, which was used as the first stage reagents, positive staining was not observed in any specimen tested (data not shown). Localization of Mar series antigens in peritoneal macrophages The immunoelectron microscopic study revealed a unique pattern of distribution of each Mar series antigen with peritoneal exudate macrophages (Figs 4 and 5). Mar 1 antigen was detected in patches on the surface membrane and also on the limiting membrane of endocytotic vacuoles in the cytoplasm (Fig. 4a). Strong positive reactivity with Mar 1 was also found on the outer coat of invaginated surface membrane and on part of the surface membrane (Fig. 5a, b). Most of the positive staining with Mar 2 was found within relatively small cytoplasmic vesicles (Fig. 4b). Some reaction products were seen on limited parts of the surface membrane (Fig. 4b), whereas, Mar 3reactive molecules were detected diffusely on the surface membrane, although the density of antigen was variable. Antigen was presented on the limiting membrane of some coated vesicles and also an invaginated membrane within the cytoplasm (Fig. 4c). The negative control using P3X supernatant or 197 mAb did not show any detectable reaction product on the surface membrane of macrophages (data not shown).

DISCUSSION In the present study, the three mAb, Mar 1, Mar 2 and Mar 3, were defined that recognized different macrophage antigens in the rats. Although OK 432-activated macrophages were used as an immunogen to produce mAb, all three reactive antigens defined with the above mAb were found in not only activated peritoneal macrophages but also residential populations, and appeared to be species specific but showed no strain specificity. Of the three mAb, Mar 1 antibody (IgGI) specifically defined the cells constituting the mononuclear phagocyte system (MPS; Van Furth et al., 1972), and thus provides a highly specific novel marker for the identification of rat MPS cells, which possess exclusively phagocytic activity. A most crucial criteria for the MPS cells among a broad spectrum of functional activities is phagocytic activity, which is expressed commonly on most of the MPS cells (Metchnikoff, 1892). Thus, Mar I antigen seems to be a common and functional molecule expressed specifically on the MPS cells. Following a comparative study of the Mar mAb with other previous mAb directed against rat macrophage populations, it was suggested that Mar 1 seems to be a unique antigen than differs from others in a variety of ways. Most of the previous mAb are directed selectively against certain populations of rat macrophages. ED 2 and ED 3, described by Dijkstra et al.

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267

Table 1. Cell populations labelled with Mar series mAb mAb

Characterization and cell type Isotype MW Staining pattern on macrophages

(MO)

Mar I

Mar 2

Mar 3

IgG I 95,000 Patchy on membrane,

IgG I 100,000

IgG2b 55,000 and 27,000 Diffuse on membrane, vesicular in cytoplasm

Vesicular in cytoplasm

vesicular in cytoplasm

Peritoneal cells Resident MO 4-day OK 432-activated MO 1-day OK 432-elicited granulocyte Blood cells Erythrocytes Granulocytes Monocytes Lymphocytes Thoracic duct lymphocytes Bone marrow cells Monocytes M0 in erythrocytic cluster Erythrocytic series Megakaryocytic series Granulocytic series Bronchial lavage M0

++ ++

++ ++

-

Splenic dendritic cells

++ ++ +

+

+

+ +

+ +

+ +

+ ++

++ +

++

+ ++ +

+

+ + stained strongly; + stained moderately; ± stained very weakly; -not stained.

11 0. _

~~~~~~~~~~~~~~~~~~~~~~~

...

(a)

-

Figure 3. Cryostat section of spleen or cell smears labelled by the immunoperoxidase technique using Mar 1. (a) Peritoneal exudate cells (4-day OK 432-induced): most of peritoneal macrophages (M4O) are strongly positive. Note the positive staining on the surface membrane and within the cytoplasm. The neutrophils (N) and lymphocytes (L) are negative. (b) Spleen: macrophages strongly stained with Mar 1 are seen in the germinal centre (GC) of a follicle (F). Magnification; (a) x 800; (b), x 240. Haematoxylin counter stain.

(1985), recognize membrane antigens of lymphoid tissue macrophages but not blood monocytes. ED I is directed against a cytoplasmic antigen of the majority of both tissue and free macrophages and blood monocytes, which mostly belong to the MPS (Dijkstra et al., 1985). However, the MW of these antigens are not identified. Ki-M2R, described by Wacker, Razun & Parwaresch (1985) selectively labels most of the MPS cells, but

not normal blood monocytes nor dendritic cells. Ku-i, described by Bodenheimer et al. (1988), shows selective reactivity with tissue macrophages and Kupffer cells but not with blood monocytes, regardless of their functional status. These mAb are distinguished from Mar I by their lack of reactivity with blood monocytes and different MW. Rumpold et al. (1982) has reported a mAb designated VEP-6 that specifically recognizes

268

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Table 2. Tissue distribution of Mar serries antigens macrophages and other non-lymphoid acciessory cell types

mAb Tissues examined

Thymus Cortex Medulla Cortico-medullary junction Hassall's corpuscle epithelium Reticulum cells Lymph node Capsule Cortex Follicle TBM Paracortex PVC endothelium Medulla Littoral cells Spleen White pulp Follicle TBM Marginal zone PALS Red pulp Peyer's patch Follicle Dome epithelium Absorptive epithelium (bottom) Liver Kupffer cells Kidney Mesangial cells Tubules Proximal Distal Lung Alveolar Interstitial Skin Langerhans' cells Dermis Thyroid gland Follicle epithelium Brain Microglia Perithelium of small blood vessel Trachea Epithelium Placenta Hofbauer cells Plexus chorioidea Epithelium + + stained strongly; + stained mo4 -

Mar 1

Mar 2

Mar 3

++ ++

± ± ± -±

++ ++ ++ +

-+

-

++

++

+

- +++

++ ++

++

++

++

++ + + ++

± + + ++

++ + + ++

++

± ++-

++ + +

++

±

+

+

-

-

++

++

++

-+

+

++ ++

++

±

++ ++

++

++

++

+

++

- +++

++

+

- + +

+

-+

+

+

derately; ± stained very weakly;

not stained

PALS, periarteriolar lymphoid shea th; TBM, tingible body macrophages.

alveolar macrophages in rats. Both OX41 and OX42, reported by Robinson et al. (1986), and R2-1 A6 and R2-2B1 by Ishii et al. (1984), recognize both macrophages and granulocytes but apparently differ from Mar 1 because of reactivity with granulocytes and different molecular sizes. Recently, Takeya, Hsiao & Takahashi (1987) reported that TRPM-3 recognizes the particular membrane antigen expressed by only restricted macrophage populations in rat lymphoid tissues but not by blood monocytes. TRPM-3 distribution on macrophages, which is restricted to the outer surface of plasma membrane, is clearly different from any of the Mar 1-3 antigens, because all the Mar 1-3 antigens are located on both surface membrane and cytoplasmic membrane structures, particularly on the limiting membrane of phagocytic small vesicles and phagosomes (Figs 4 and 5). The molecular nature of TRPM-3 antigen is not known. Taken together, there is no reported mAb that can recognize common antigen of macrophages and thus cover specifically the MPS in rats except for Mar l described in the present study. On the other hand, it was shown that Mar 2 and Mar 3 antigens appear to be different from Mar 1 antigen in tissue distribution and molecular nature (Tables 1 and 2). There exists a considerable overlap between the distribution patterns of the former two antigens, because they were distributed on both free macrophages populations and fixed endocytotic epithelia, e.g. the epithelium of thyroid follicles or choroid plexus (Tables 1 and 2). Mar 1 antigen was located exclusively on the various free macrophage populations which constitute the MPS. In one aspect of the distribution pattern, Mar 3 antigen seems to partially resemble shared antigens between granulocytes and macrophages, which are defined by R2-2B1 (Ishii et al., 1984) or OX41 and OX42 (Robinson et al., 1986), since all of these antigens were distributed not only on the macrophage-granulocyte series, but also in follicular dendritic cells or endothelium of the PCV in the lymphoid tissues. However, Mar 3 antigen is quite different from all of these antigens, because of the difference in molecular natures and in the distribution patterns in the non-lymphoid tissues, including fixed endocytotic cells. Table 2 shows that Mar 3 mAb can recognize two antigen molecules of 55,000 MW and 27,000 MW, which are expressed on the membranous structures of both cell populations constituting the MPS and cells exhibiting endocytotic activity; the later population contains not only granulocytes from blood and bone marrow but also cells from non-lymphoid tissues, e.g. tubular epithelium of kidney or thyroid follicular epithelium. The immunoelectron microscopic study revealed that the cellular distribution of Mar 3 antigen was somewhat different from that of Mar 2 in density and pattern, because the distribution of Mar 3 antigen on peritoneal macrophages was more dense and diffuse on the surface membrane and cytoplasmic vesicles than that of Mar 2, which is selectively restricted to the cytoplasmic vesicles (Fig. 4). It is noteworthy that all of the Mar series mAb weakly stained splenic dendritic cells, which are characterized by their accessory cell function in the immune response and the expression of Ia antigen molecules on their cell surfaces (Table 2). The very low staining intensity of splenic dendritic cells appears to correlate with the finding that dendritic cells show no or extremely low endocytic activity (Steinman et a., 1980). On the contrary, splenic macrophages with strong phagocytic activity were stained with these mAb far more strongly than dendritic cells. Thus, this implies that the degree of expression of antigen

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Figure 4. Immunoelectron micrographs of peritoneal macrophages stained with Mar series mAb. (a) Mar 1: strong positive immunocytochemical reactivity is located on part of the surface membrane and the limiting membrane of endocytotic vacuoles in the cytoplasm. (b) Mar 2: most of the positive reactivity is located within the cytoplasm of the macrophages, particularly on the limiting membrane of small vesicles. Some reaction products are seen on limited parts of the surface membrane (arrowed). (c) Mar 3: Mar 3 reaction molecules are detected diffusely on the cell surface and the limiting membrane of some coated vesicles (arrowed). Note the presence of strong positive reactivity on the invaginated surface membrane. (a-e) Magnification x 12,000.

molecules defined with these mAb may reflect the intensity of phagocytic activity. Immunohistochemical staining using MRC OX6 showed that most of the splenic dendritic cells are strongly positive on Ia antigen (McMaster & Williams, 1979). The presence of the Mar series antigens on other dendritic cells, e.g. the follicular dendritic cells or the interdigitating cells in the lymph node (Witmer & Steinman, 1984), is not clear in the present study. It is of interest that dendritic cell populations in the lymph node may gain the expression of phagocytic antigen molecules during their differentiation process, as defined by the Mar series mAb in splenic dendritic cells, and then are endowed with a weak phagocytotic activity. In an attempt to elucidate the functional activity of the Mar series antigens on the macrophage surface, Mar 1-3 mAb were recently tested for inhibition of specific macrophage function, especially phagocytotic activity in vitro. Pretreatment of peritoneal macrophages with an appropriate dose of either Mar 1 or

Mar 3 at 4G for I hr resulted in a marked reduction of the uptake of latex particles 1-3 hr after the incubation with latex particles at 37° (A. Yamashita, Y. Hattori, M. Kotani and M. Inada, manuscript in preparation). The degree of the uptake inhibition by Mar 3 pretreatment was much higher than that of Mar 1. No definite inhibitory activity was found in Mar 2 pretreatment. On the other hand, there was no difference in the degree of uptake inhibition by Mar 3 or Mar 1 pretreatment between IgG- or C3opsonized sheep red blood cells and non-opsonized cells. These findings indicate that Mar 3 and Mar 1 mAb can preferentially block or abolish the endocytotic activity of macrophages, and thus recognize phagocytosis-associated molecules on macrophages, particularly in the process of non-professional phagocytosis in vitro, which is not mediated by C3b receptor or Fc receptor. Of the three mAb, Mar 1 mAb seems to recognize a common and functional molecule expressed specifically in the cells

A. Yamashita et al.

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Figure 5. Distribution pattern of Mar 1- reactive molecules on peritoneal macrophages. (a) The strong positive reactivity is observed along the outer surface of invaginated membrane (arrowed) and on part of the cell surface as a patch-like staining. Note the variability of the Mar 1 antigen distribution. (a) x 15,000; (b) x 31,000.

constituting the MPS, and would be useful for the identification of the MPS cells, the assessment of functional capability and in vivo differentiation processes of the macrophage populations.

ACKNOWLEDGMENTS The authors wish to express their thanks to the late Professor B. Morris, Department of Immunology, the John Curtin School of Medical Research, Canberra, Australia, for his comments and reviews of the manuscript.

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