Immunobiol., vol. 182, pp. 88-99 (1990)
Department of Cell Biology, Division Histology, Medical Faculty, Vrije Universiteit, Amsterdam, The Netherlands
Characterization and Expression of the Antigen Present on Resident Rat Macrophages Recognized by Monoclonal Antibody ED2 ELLIS BARBE, JAN G.
M. C. DAMOISEAUX, ED A. Dopp, and CHRISTINE D.
DI]KSTRA
Received April 12, 1990 . Accepted in Revised Form October 31, 1990
Abstract Because of the absence of a specific marker for labeling resident macrophages in the rat, there is almost no information available regarding the properties of individual resident macrophages in different organs. The recently described and in our laboratory developed mAb ED2, has been shown to exclusively recognize resident macrophages. The present study examines expression, function and structure of the ED2 antigen to obtain more information about the marker and therefore, more information about resident macrophages. In earlier studies, the expression of ED2 could not be induced by a range of macrophage stimulating factors under non-adherent culture conditions. We show a highly inducible expression of the ED2 antigen under adhering, non proliferating conditions as well as in long-term bone marrow cultures. ED2 appears to recognize a surface protein on resident macrophages consisting of three protein chains of 175, 160, and 95 kDa.
Introduction Precursors of macrophages originate in the bone marrow, differentiate into circulating monocytes which leave the peripheral blood for the tissue compartments (1). Once they have entered tissues they differentiate further into macrophages and often adopt a characteristic morphology, depending on their localization and further migration (2). Normal tissue macrophages become a resident character in the absence of an endogenous inflammatory stimulus (1). Resident macrophages are characterized by a specific pattern of endogenous peroxidatic activity, located in the nuclear envelope and rough endoplasmatic reticulum (3). In many normal tissues, resident macrophages provide a first line of defense against injury and infection (2). The local functions of resident macrophages in different tissues are still obscure, but recent studies brought Abbreviations: BP = bacterial plastic; FCS = fetal calf serum; NFCS = newbornlfetal calf serum; FN = fibronectin; ECM = extra cellular matrix; LCM = L-929 cell conditioned medium; LTBC = long-term bone marrow culture; mAb = monoclonal antibody; PBS = phosphate buffered saline.
The ED2 macrophage differentiation antigen . 89
to light novel properties. Resident macrophages in various tissues have much in common, but they express regional differences according to their unique micro-environments (2). Stromal resident macrophages in bone marrow make close contact with developing haemopoietic cells. These macrophages may play an important role in trophic and regulatory interactions with adjacent haemopoietic cells (4, 5). It is postulated that stromal macrophages in the thymus play a crucial role in several stages of T cell differentiation, including T cell receptor diversification and selection of self H-2 restricted T cells (6-8). Functional properties of resident red-pulp macrophages in the spleen and Kupffer cells, resident macrophages in the liver, are quite similar (9). These macrophages express several membrane receptors involved in clearance of senescent blood cells (2). Resident peritoneal macrophages have many typical endocytic and secretory properties (9). In mice there are several methods to distinguish resident macrophages (10). For example the mAb F4/80 recognizes a membrane antigen on mouse macrophages from different sites but not on closely related haemopoietic cells (11, 12). This mAb is often used to characterize resident macrophages although it also recognizes monocytes. In the rat a protein analogue of the F4/80 antigen is found (13). Previously we described the mAb ED2, recognizing a membrane antigen present predominantly on fixed tissue macrophages of rats (14). In different organs, ED2 positive cells with extensive cell processes are present which seem to be strongly fixed into the connective tissue. The bone marrow macrophages in erythropoietic clusters, the thymic cortical macrophages, liver Kupffer cells, and splenic red-pulp macrophages are all ED2 positive (14). Monocytes, dendritic cells, lymphocytes and granulocytes are negative for ED2. No cell types other than those belonging to the mononuclear phagocyte system are positive for ED2 (14). As detected by simultaneous demonstration of endogenous peroxidase activity on the ultrastructural level, ED2 expression was correlated with resident macrophages (15). In the present study, the in vitro expression, function, and structure of the antigen recognized by the mAb ED2 were examined. We examined the ED2 expression in several culture systems and in particular, the influence of reticular components, extra cellular matrix proteins (ECM) as well as reticular cells. We show that macrophage differentiation is enhanced under adhering, non proliferating conditions as well as in long-term bone marrow cultures (L TBC).
Materials and Methods Animals
Wistar rats were obtained from HSD/CPB, Zeist, The Netherlands, and kept under routine laboratory conditions.
90 . ELLIS BARBE, JAN G. M. C. DAMOISEAUX, ED A. Dopp, and CHRISTINE D. DI]KSTRA Bone marrow culture Bone marrow was obtained from young adult rats. The femora were excised aseptically, the end of the bones were removed, and the marrow was flushed out, using 5 ml of RPMI-1640 medium (Gibco, Uxbridge, U.K.) expelled from a 10 ml syringe through a 20-gauge needle. The released cells were washed by centrifugation and resuspended in culture medium consisting of: RPMI-1640 medium, 2 mM glutamin, 100 Ulml penicillin, and 100 glml streptomycin, supplemented with 20 % fetal calf serum (FCS) (Hyclone Laboratories Inc., UT, USA). The macrophage precursor cells were enriched by culturing the total bone marrow fraction for 3 days in 6 plastic culture flasks of 75 cm2 (each flask contained approximately 7.5 x 106 cells in 15 ml culture medium). The culture medium was supplemented with 20 % L929 cell conditioned medium (LCM) as a source of M-CSF (16). The non-adherent cell fraction containing the macrophage precursor cells, was harvested after 3 days. The in vitro expression of ED2 was studied by culturing macrophage precursor cells under different culture conditions. Non-adherent and adherent culture conditions were tested by culturing the macrophage precursor cells in teflon bags (17) or on covers lips placed in 24-well culture trays (16). The culture medium was differentially supplemented with 20 % LCM. The precursor cells were cultured in a concentration of approximately 106 cells/ml culture medium. Several different adherent culture conditions were tested by culturing the precursor cells on covers lips coated with poly-L-lysin (10 f!g/well) (Sigma, St. Louis, MO, USA», fibronectin (FN) (5 f!g/well) (Boehringer, Mannheim, Germany), or ECM. The ECM was produced by cells of a rat LTBC (see below). The adherent layer of a LTBC is capable of producing soluble factors which influence haemopoietic proliferation and/or differentiation and contain a variety of adhesion factors such as fibronectin, laminin, and type IV collagen (18). The supernatant of the LTBC was used for coating. For studying the ED2 positive macrophage population in bone marrow stroma, L TBC were used (18). Total bone marrow was cultured on covers lips placed in a 24-well tissue culture tray. After centrifugation, the total bone marrow fraction was resuspended in 75 ml culture medium with addition of 10 % newbornlfetal calf serum (NFCS) instead of FCS, divided over 3 culture trays containing coverslips, and cultured for several weeks. All experiments were done under optimal culture conditions at 37°C and 95 % air/5 % CO 2 in a humidified incubator. After culturing, cells were harvested from teflon bags and cytocentrifuged onto glass-slides. Glass-slides and coverslips were differentially stained for ED1, ED2 and acid phosphatase. Differential cell counts (100 cells) were performed on each preparation. Cells of the mononuclear phagocyte lineage were characterized by morphology, acid phosphatase activity and ED1 staining. All experiments were repeated at least three times. fmm unohistochemistry The following mAbs were used: ED1, a highly specific marker for cells of the mononuclear phagocyte lineage (14); ED2, a marker for a subpopulation of macrophages (14). As conjugate a rabbit-anti-mouse IgG/peroxidase (DAKOpatts, Copenhagen, Denmark) was used. Coverslips were rinsed in saline with 0.01 M phosphate buffered saline (PBS) pH 7.4 whereas cytospins were allowed to dry overnight. Coverslips and cytospins were fixed in aceton containing 2.5 % formol. After fixation the samples were rinsed with PBS and incubated with the mAbs. MAbs were diluted in PBS with 2.5 % bovine serum albumine (BSA) and used in optimal concentrations. After incubation with the first antibody for 30 min, the samples were rinsed thoroughly in PBS, incubated in conjugate for 30 min, washed again thoroughly in PBS, and stained for peroxidase activity with 0.5 mg/ml 3,3' -diaminobenzidinetetrahydrochloride (Sigma) in 0.05 M Tris-HCI buffer, pH 7.6 containing 0.01 % H 2 0 2 for 10 min. After rinsing in PBS the samples were slightly counterstained with haematoxylin, dehydrated and mounted in Entellan (Merck, Darmstadt, Germany). Control samples were incubated in PBS-BSA in the first step, with conjugate in the second step and examined for non-specific staining. Acid phosphatase activity was demonstrated according to the method of BURSTONE (19).
The ED2 macrophage differentiation antigen . 91 Radiolabeling and immunoprecipitation Peritoneal cells were surface labeled with Na l2S I (Amersham International, Amersham, U.K.) using lactoperoxidase (Sigma) and H 2 0 2 (20). Radiolabeled cells were lysed with 1 % Nonidet P-40 in 0.01 M triethanolamine-HCl, pH 7.8, 0.15M NaCI. Proteolysis inhibitors used are 1 mM phenyl methyl sulfonyl fluoride, 1 fAg/mlleupeptin, 1 fAg/ml pepstatin, 1 fAg/ml antipain. Nuclear debris was removed from the lysates by centrifugation at 15,000 x g for 5 min at 4°C. Preclearing of the lysates was done with normal mouse serum for 30 min succeeded by protein G Sepharose (Pharmacia LKB, Uppsala, Sweden) for another 30 min. Precleared lysates were incubated for 1 h with the ED2 or control mAb, succeeded by protein G Sepharose for 30 min. Immunoprecipitates were removed from the Iysates by centrifugation at 15,000 x g and were washed four times in 0.05 M Tris-HCl, pH 7.5, 0.5 % NP40, 5 mM EDTA, O.l5M NaCI. Electrophoresis and autoradiography SDS polyacrylamide gel electrophoresis was carried out on 10% acrylamide vertical slab gels according to LAEMMLI (20). Immunoprecipitates were dissolved by boiling for 5 min in electrophoresis sample buffer. The gels were run under reducing conditions, fixed, dried, and autoradiography was performed at -70°C using Kodak XAR-5 film. Adhesion assay To examine the adhesive capabilities of the ED2 antigen the adhesion assay modified after ROSEN et al. (21) was performed. Peritoneal cells were suspended in RPMI-1640 medium containing 1 % BSA (Organon Technika, Boxtel, The Netherlands), and plated at a density of 1 x 105 or 2 x 105 cells/well in 96-well plates of bacterial plastic (BP) (Greiner, Alphen aan de Rijn, The Netherlands). The plates were differentially coated with FN, ECM, or poly-L-Iysine as described above. After incubation for 30 min at 3rC, plates were washed three times in PBS and adherent cells were fixed with methanol. After staining with 0.2 % Toluidine Blue solution for 10 min, plates were washed in water, dried and the retained dye was solubilized in methanol. Stain was quantified by measuring absorbance at 620 nm in an automatic plate reader (Titertek Multiscan MCC/340). In antibody inhibition experiments, peritoneal cells were incubated with 10 fAl ascites of the mAb during 15 min at 4°C. Antibody excess was removed by extensive washing prior to the adhesion assay.
Results
The influence of adherent conditions on ED2 expression in bone marrow cultures To study the ED2 expression during adherent and non-adherent conditions, macrophage precursor cells were cultured on coverslips and in teflon bags. About 4 x 10 7 cells, containing macrophage precursors, could be h~rvested after culturing the total bone marrow on culture plastic for 3 days. Approximately 50 % of the harvested non-adherent fraction stained positive for EDl and acid phosphatase. About 3 % of the cells stained positive for ED2. When the harvested cells were cultured onto glass coverslips, almost all cells adhered after 3 days. This indicates that there was no selection by differences in adhesion. The percentage of mononuclear phagocytes increased from 50 % on day o via 80 % on day 7, 90 % on day 10 to almost 100 % in course of two
92 . ELLIS BARBE, JAN G. M. C. DAMOISEAUX, ED A. Dopp, and CHRISTINE D. DI]KSTRA
Days 01 culturing
Figure 1. Influence of adherent conditions on ED2 expression in bone marrow cultures. Bone marrow cells were cultured on glass-coverslips and in teflon bags. The culture medium was supplemented with 20 % FCS. The data represent mean percentages ± SD of macrophages positive for ED2.
weeks in both teflon and coverslip cultures. The percentage of macrophages positive for ED2 on coverslips increased from 3 to 90 % in course of one week and decreased thereafter to 25 % on day 14 of culturing (Fig. 1). There was however a variation in the level of ED2 expression observed. During culturing a maximum of 75 % of the cells stained weakly for ED2, whereas a maximum of only 15 % stained strongly for ED2. 100
% ED2 80
D ~
gil. weak 51
gil. strong st
60
40
20
0
Figure 2. Influence of LCM on ED2 expression in bone marrow cultures. Bone marrow cells were cultured on coverslips and in teflon bags. The culture medium was supplemented with 20 % FCS and 20 % LCM. The data represent mean percentages ± SD of macrophages positive for ED2.
The ED2 macrophage differentiation antigen . 93
In teflon, no increase of ED2 expression could be noticed. The ED2 positive macrophages remained between 3 % and 6 % during 14 days of culturing (Fig. 1).
The influence of LeM on ED2 expression Macrophage precursor cells were cultured in culture medium supplemented with 20 % LCM, on coverslips and in teflon bags. After 3 days of culturing in teflon and coverslip cultures, 80 % of the cells became positive for EDl and stained positive for acid phosphatase. On day 7, almost all cells were macrophages. On coverslips the percentage macrophages positive for ED2 increased from 3 to 62 % in course of 14 days, whereas the percentage of ED2 positive macrophages from the teflon culture, remained at a low level during culture time (Fig. 2). Addition of LCM to the culture medium stimulated macrophage proliferation, resulting in a high number of macrophage precursor cells. Culturing in the presence of LCM again resulted in a variable level of ED2 expression (Fig. 2).
ED2 expression in the presence of stromal components In different organs, adherent resident macrophages with extensive cell processes can be found which seem to be strongly fixed into connective tissue. To study the ED2 expression in relation to adherence to stromal components, bone marrow precursor cells were cultured on coverslips coated with several materials. Coating coverslips with FN or ECM stimulated the proliferation resulting in a higher number of macrophages than the number of macrophages on uncoated coverslips. Compared to glass, the
100
%
ED2
0
80
glas weak 5t
glas strong 51
EeM
F N
60
poly-L-Iysln
40
20
0
Days 01 cultUring
Figure 3. ED2 expression in a bone marrow culture in the presence of several adhesion components. Macrophages were cultured on coverslips differentially coated with FN, ECM, or poly-L-Iysin. The data represent mean percentages ± SD of macrophages positive for ED2.
94 . ELLIS BARBE, JAN G. M. C. DAMOISEAUX, ED A. Dopp, and CHRISTINE D. DI]KSTRA
presence of poly-L-lysin, FN, or ECM did not result in an extensive increase of ED2 expression (Fig. 3). To determine macrophage differentiation in LTBC, freshly isolated total bone marrow was cultured on coverslips. The culture medium was supplemented with 10 % NFCS. After 14 days of culture, a stromal monolayer was produced. Approximately 70 % of the cells stained positive for EDl and acid phosphatase and approximately 35 % of the monolayer cells stained positive for ED2.
Heterogeneity of the cultured cells The macrophages cultured in the presence of LCM showed a round morphology with a round oval excentric nucleus and a large cytoplasm fraction (Fig. 4a), whereas macrophages cultured in the absence of LCM had extensive cell processes with less cytoplasm (Fig. 4b). The cells that stained positive for ED2 also had predominantly extensive cell processes (Fig. 4c). On cytospins of the macrophages from the teflon bags, different
•••• B
! c ·
, 0
Figure 4. Morphology of macrophages cultured on coverslips. A: macrophages cultured for 3 days in the presence of 20 % LCM, stained for EDl, magnification 400x. B: macrophages cultured for 3 days in the absence of LCM, stained for EDl, magnification lOOOx. C: macrophages cultured for 7 days in absence of LCM, stained for ED2, magnification lOOOx. D: LTBC of 3 weeks, stained for ED2, shows a mixture of ED2 positive macrophages with extensive processes and ED2 negative fibroblastic cells, magnification 400x.
The ED2 macrophage differentiation antigen . 95
1
200
2
~
116 ~ 96~
68~
Figure 5. Analysis by immunoprecipitation of the antigens recognized by ED2. Resident peritoneal cells were surface labeled with 125 1, solubilized in detergent and immunoprecipitated with an antibody and Protein G Sepharose. 1mmunoprecipitates were subjected to SDS 10% PAGE under reducing conditions followed by autoradiography. Lane 1: ED2. Lane 2: irrelevant antibody control. The position of the molecular weight markers is given on the left side of the figure.
morphology could not be noticed in presence or absence of LCM. Except in morphology, a clear distinction could be made under all circumstances in the extent of ED2 expression. There was no gradual transition in expression, but only weak or strong expression, or no expression at all. In LTBC (Fig. 4d) the cells which did not stain positive for EDl had a fibroblastic morphology with a large oval nucleus with several conspicious nucleoli and a large cytoplasm fraction. The cells which stained positive for ED2 in the LTBC could be divided into two groups. One group seemed to adhere strongly to the glass, had extensive cell processes, made contact with surrounding cells and was part of the monolayer. The other group had a round appearance and adhered slightly to the monolayer or not at all. The cells which stained negative for ED2 were either fibroblastic cells or small round macrophages with a small round nucleus and a small cytoplasm fraction.
Molecular weight and adhesion assay The results of immunoprecipitation using the ED2 mAb and the Na 125 I_ labeled peritoneal macrophage lysates, showed that the ED2 mAb
96 . ELLIS BARBE, JAN G. M. C. DAMOISEAUX, ED A. Dopp, and CHRISTINE D. DIjKSTRA
immunoprecipitated three protein chains of Mr 175,000, 160,000, and 95,000 under reducing conditions (Fig. 5). This molecular weight has characteristics of the molecular weights of the integrins, a family of adhesive receptors (22). In order to test whether the ED2 antigen is a cell surface protein with adhesive properties, we studied the adhesion of peritoneal cells, which contain 70 % mononuclear phagocytes and approximately 30 % ED2 positive cells (14). The ED2 mAb was not able to inhibit macrophage adhesion (Table 1). To investigate the role of several adhering substrates, the cells were differentially coated with FN, ECM, or poly-L-Iysin. Coating with ECM resulted in a significant 80 % decrease in adherence compared to plastic. Poly-L-Iysin and FN coating resulted in a 50 % increase in adherence. Incubation of the peritoneal cells with the ED2 mAb did not result in adherence inhibition to these substrates (Table 1). Discussion The use of liquid bone marrow cultures offers an in vitro approach for studying macrophage differentiation (16, 18,23). The ED2 mAb recognizes a differentiation antigen on resident macrophages and is not expressed on monocytes and recently recruited inflammatory macrophages (14, 15,24). In freshly isolated bone marrow, almost 100 % of the typical mature macrophages is ED2 positive, whereas precursors are ED2 negative (23). In earlier studies, the expression of ED2 could not be induced under different non adherent culture conditions. In this study however, the ED2 antigen was inducible on bone marrow derived macrophages if the culture system provided the appropriate conditions for differentiation. The ED2 expression appeared to be restricted to environments where adhesive components are available. In vitro, no ED2 positive cells were observed when teflon bags were used for culturing the precursor cells. When precursors were cultured on materials to which mature macrophages could adhere, glass, FN, ECM, or polyL-lysin, ED2 was highly inducible. When LCM is added to cultures, Table 1. Adhesion assay
Bacterial Plastic Poly-L-lysin Extra-Cellular Matrix Fibronectin
Control
Control mAb
ED2 mAb
241 367 SO 363
215 ± 291 ± O± 261 ±
228 321 SO 313
± ± ± ±
10 20 30 10
10 10 0 20
± ± ± ±
10 10 10 60
Adhesion of peritoneal macrophages to plastic differentially coated with Poly-L-lysin, ECM, or Fibronectin. About 105 cells per well were plated in 96-well plates of bacterial plastic. Results are presented as mean absorbance at 620 nm ± SD (x 103 ) of triplicate experiments.
The ED2 macrophage differentiation antigen . 97
macrophage proliferation is stimulated, resulting in a higher number of mononuclear phagocytes but a relatively lower number of mature macrophages (16). This explains the fact that when LCM is added to the culture a lower number ED2 positive macrophages is observed. Several in vivo studies support the idea that adhesion is necessary to induce ED2 expression. After macrophage elimination in spleen using liposome-encapsulated dichloromethylene diphosphonate, it takes about 8 days before the red-pulp is repopulated by ED1 positive macrophages. Only after another week do these macrophages start to express ED2 (25). Also in ontogenetic development of the lymphoid organs the ED2 expression is observed about 1 to 2 weeks after the first macrophages have populated these tissues (26, 27). Apparently the ED1 positive macrophages require about a week to become ED2 positive after entry into a tissue, and thus need adherence to connective tissue. However, we showed that the ED2 antigen is also expressed on non-adherent cells in LTBC, that is after contact with the bone marrow fibroblastic cells. Also in vivo about 50 % of the free resident peritoneal macrophages express ED2 (14, 24). This indicates that interaction with adhesive components is necessary to induce ED2, but not for maintenance of the antigen expression. Macrophage adhesion plays an important role in a variety of processes including phagocytosis, inflammation, immunity and arteriosclerosis (21). Selective interaction with adhesive components, like FN, laminin, and vitronectin depends on specific plasma membrane receptors (28-30). ECM, consisting of a variety of proteins including FN, laminin, type IV collagen, and several growth factors, plays an important role in in vitro proliferation and differentiation of haemopoietic cells (31). Our study shows that macrophages exhibit differential affinity for these substrates as studied in the adhesion assay. They have high affinity for FN and poly-L-Iysin, but low affinity for ECM, although this mixture also contains FN. As stated above, many different substrate-specific plasma membrane receptors might be responsible for this differential affinity. In spite of this difference in affinity, we showed in the culture system that after three days nearly all cells adhered to the same extend to the four substrates used. Furthermore, the presence of poly-L-Iysin, FN, or ECM did not result in a difference in the percentage ED2 positive macrophages. However, when we take into account the higher proliferation rate on FN and in the presence of LCM, these two substrates might accelerate the induction of ED2 expression and therefore differentiation. The molecular weight of the ED2 antigen is suggestive for being a member of the integrin superfamily of adhesion molecules. Especially the 95 kDa subunit points in the direction of the LFA-l family, which is specific for leukocytes (22). However, the tissue distribution of the ED2 antigen is not in agreement with any of the members of the LFA-family (14, 22). Furthermore, we showed that the mAb ED2 was not able to inhibit adhesion.
98 . ELLIS BARBf:, JAN G. M. C. DAMOISEAUX, ED A. Dopp, and CHRISTINE D. DIJKSTRA
Taken together, our results show that ED2 is a very usefull tool for in vitro labeling of resident macrophages in rat. The in vivo functional relevance of the ED2 antigen needs further investigation.
References 1. VAN FURTH, R. 1982. Current view on the mononuclear phagocyte system. Immunobiol. 161: 178. 2. GORDON,S., S. KESHAV, and L. P. CHUNG. 1988. Mononuclear phagocytes: tissue distribution and functional heterogeneity. Curr. Opin. Immunol. 1: 26. 3. BEE LEN, R. H. J., D. M. BROEKHUIS-FLUITSMA, and E. C. M. HOEFSMIT. 1979. Peroxidatic activity in mononuclear phagocytes. Ultramicroscopy 3: 129. 4. CROCKER, P. R., L. MORRIS, and S. GORDON. 1988. Novel cell-surface adhesion receptors involved in interactions between stromal macrophages and haematopoietic cells. J. Cell Sci. (5) 9: 185. 5. LEE, S. H., P. R. CROCKER, S. WESTABY, N. KEY, D. Y. MASON, S. GORDON, and D. J. WEATHERALL. 1988. Isolation and immunocytochemical characterization of human bone marrow stromal macrophages in haemopoietic clusters. J. Exp. Med. 168: 1193. 6. BELLER, D. I., and E. R. UNANUE. 1978. Thymic macrophages modulate one stage of T cell differentiation in vitro. J. Immunol. 121: 1861. 7. BELLER, D. I., and E. R. UNANUE. 1980. Ia antigens and antigen presenting function of thymic macrophages. J. Immunol. 124: 1426. 8. ZEIRA, M., and R. GALLILY. 1988. Interaction between thymocytes and thymus-derived macrophages. Cell. Immunol. 117: 277. 9. NUSRAT, A. R., S. D. WRIGHT, A. A. ADEREM, R. M. STEINMAN, and Z. A. COHN. 1988. Properties of isolated red pulp macrophages from mouse spleen. J. Exp. Med. 168: 1505. 10. MELNICOFF, J., P. S. MORAHAN, B. D. JENSEN, E. W. BRESLIN, and P. K. HORAN. 1988. In vivo labeling of resident peritoneal macrophages. J. Leukocyte BioI. 43: 387. 11. AUSTYN,j. M., and S. GORDON. 1981. F4/80, a monoclonal antibody directed specifically against the mouse macrophage. Eur. j. Immunol. 11: 805. 12. HUME, D. A., A. P. ROBINSON, G. G. MACPHERSON, and S. GORDON. 1983. The mononuclear phagocyte system of the mouse defined by immunohistochemicallocalization of antigen F4/80. j. Exp. Med. 158: 1522. 13. STARKEY, P. M., L. TURLEY, and S. GORDON. 1987. The mouse macrophage-specific glycoprotein defined by monoclonal antibody F4/80; characterization, biosynthesis and demonstration of a rat analogue. Immunology 60: 117. 14. DIJKSTRA, C. D., E. A. Dopp, P. jOLING, and G. KRAAL. 1985. The heterogeneity of mononuclear phagocytes in lymphoid organs; distinct macrophage subpopulations in the rat recognized by monoclonal antibodies EDl, ED2 and ED3. Immunology 54: 589. 15. BEELEN, R. H. J., I. L. EESTERMANS, E. A. Dopp, and C. D. DIJKSTRA. 1987. Monoclonal antibodies EDl, ED2, and ED3 against rat macrophages: expression of recognized antigens in different stages of differentiation. Transplant. Proc. XIX 3: 3166. 16. BOLTz-NITULESCU, G., C. WILTSCHKE, C. HOLZINGER, A. FELLINGER, O. SCHEINER, A. GESSL, and O. FORSTER. 1987. Differentiation of rat bone marrow cells into macrophages under the influence of mouse L929 cell supernatant. J. Leukocyte BioI. 41: 83. 17. VAN DERMEER, J. W. M., J. S. VAN DE GEVEL, I. ELZENGA-CLAASSEN, and R. VAN FURTH. 1979. Suspension cultures of mononuclear phagocytes in the teflon culture bag. Cell. Immunol. 42: 208. 18. SULLIVAN, A. K., D. CLAXTON, G. SHEMANTHEK, and H. WANG. 1989. Cellular composition of rat bone marrow stroma. Lab. Invest. 60: 667. 19. BURSTONE, M. S. 1958. Histochemical comparison of naphtol-phosphatases for the demonstration of phosphatases. J. Natl. Cancer Inst. 20: 601.
The ED2 macrophage differentiation antigen . 99 20. LAEMMLI, U. K. 1970. Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature 227: 680. 21. ROSEN, H., and S. GORDON. 1987. Monoclonal antibody to the murine type 3 complement receptor inhibits adhesion of myelomonocytic cells in vitro and inflammatory cell recruitment in vivo. J. Exp. Med. 166: 1685. 22. HYNES, R. O. 1987. Integrins: a family of cell surface receptors. Cell 48: 549. 23. DA~10ISEAUX, J. G. M. c., E. A. Dopp, R. H. J. BEELEN, and C. D. DIjKSTRA. 1989. Rat bone marrow and monocyte cultures: influence of culture time and lymphokines on the expression of macrophage differentiation antigens. J. Leukocyte BioI. 46: 246. 24. DAMOISEAUX, J. G. M. c., E. A. Dopp, J. J. NEEFjEs, R. H. J. BEELEN, and C. D. DIJKSTRA. 1989. Heterogeneity of macrophages in the rat evidenced by variability in determinants: two new anti-rat macrophage antibodies against a heterodimer of 160 and 95 kd (CDllICDI8). J. Leukocyte BioI. 46: 556. 25. VAN ROOIJEN, N., N. KORS, M. VAN DER ENDE, and C. D. DIjKSTRA. 1990. Depletion and repopulation of macrophages in spleen and liver of rat after intravenous treatment with liposome-encapsulated dichloromethylene diphosphonate. Cell Tissue Res. 260: 215. 26. VAN REEs, E. P., E. A. Dopp, C. D. DIjKSTRA, and T. SMINIA. 1985. The postnatal development of cell populations in the rat popliteal lymph node. An immunohistochemical study. Cell. Tissue Res. 242: 391. 27. VAN REES, E. P., C. D. DIjKSTRA, M. B. VAN DER ENDE, E. M. lANSE, and T. SMINIA. 1988. The ontogenetic development of macrophage subpopulations and Ia positive non-lymphoid cells in gut associated lymphoid tissue of the rat. Immunology 63: 79. 28. IGNOTZ, R. A., J. HEINO, and J. MASSAGUE. 1989. Regulation of cell adhesion receptors by transforming growth factor-~. J. BioI. Chern. 264: 389. 29. HUARD, T. K., H. L. MALINOFF, and M. S. WICHA. 1986. Macrophages express a plasma membrane receptor for basement membrane laminin. Am. J. Pathol. 123: 365. 30. GARCIA-PARDO, A., O. C. FERREIRA, J. VALINSKY, and C. BIANCO. 1989. Fibronectin receptors of mononuclear phagocytes: binding characteristics and biochemical isolation. Exp. Cell Res. 181: 420. 31. CAMBEL, A., M. S. WICHA, and M. LONG. 1985. Extracellular matrix promotes the growth and differentiation of murine hematopoietic cells in vitro. J. Clin. Invest. 75: 2085. Dr. J. G. M. C. DAMOISEAUX, Department of Cell Biology, Division of Histology, Medical Faculty, Vrije Universiteit, Van de Boechorststraat 7, 1081 BT Amsterdam, The Netherlands
Department of Cell Biology, Division Histology, Medical Faculty, Vrije Universiteit, Amsterdam, The Netherlands
Characterization and Expression of the Antigen Present on Resident Rat Macrophages Recognized by Monoclonal Antibody ED2 ELLIS BARBE, JAN G.
M. C. DAMOISEAUX, ED A. Dopp, and CHRISTINE D.
DI]KSTRA
Received April 12, 1990 . Accepted in Revised Form October 31, 1990
Abstract Because of the absence of a specific marker for labeling resident macrophages in the rat, there is almost no information available regarding the properties of individual resident macrophages in different organs. The recently described and in our laboratory developed mAb ED2, has been shown to exclusively recognize resident macrophages. The present study examines expression, function and structure of the ED2 antigen to obtain more information about the marker and therefore, more information about resident macrophages. In earlier studies, the expression of ED2 could not be induced by a range of macrophage stimulating factors under non-adherent culture conditions. We show a highly inducible expression of the ED2 antigen under adhering, non proliferating conditions as well as in long-term bone marrow cultures. ED2 appears to recognize a surface protein on resident macrophages consisting of three protein chains of 175, 160, and 95 kDa.
Introduction Precursors of macrophages originate in the bone marrow, differentiate into circulating monocytes which leave the peripheral blood for the tissue compartments (1). Once they have entered tissues they differentiate further into macrophages and often adopt a characteristic morphology, depending on their localization and further migration (2). Normal tissue macrophages become a resident character in the absence of an endogenous inflammatory stimulus (1). Resident macrophages are characterized by a specific pattern of endogenous peroxidatic activity, located in the nuclear envelope and rough endoplasmatic reticulum (3). In many normal tissues, resident macrophages provide a first line of defense against injury and infection (2). The local functions of resident macrophages in different tissues are still obscure, but recent studies brought Abbreviations: BP = bacterial plastic; FCS = fetal calf serum; NFCS = newbornlfetal calf serum; FN = fibronectin; ECM = extra cellular matrix; LCM = L-929 cell conditioned medium; LTBC = long-term bone marrow culture; mAb = monoclonal antibody; PBS = phosphate buffered saline.
The ED2 macrophage differentiation antigen . 89
to light novel properties. Resident macrophages in various tissues have much in common, but they express regional differences according to their unique micro-environments (2). Stromal resident macrophages in bone marrow make close contact with developing haemopoietic cells. These macrophages may play an important role in trophic and regulatory interactions with adjacent haemopoietic cells (4, 5). It is postulated that stromal macrophages in the thymus play a crucial role in several stages of T cell differentiation, including T cell receptor diversification and selection of self H-2 restricted T cells (6-8). Functional properties of resident red-pulp macrophages in the spleen and Kupffer cells, resident macrophages in the liver, are quite similar (9). These macrophages express several membrane receptors involved in clearance of senescent blood cells (2). Resident peritoneal macrophages have many typical endocytic and secretory properties (9). In mice there are several methods to distinguish resident macrophages (10). For example the mAb F4/80 recognizes a membrane antigen on mouse macrophages from different sites but not on closely related haemopoietic cells (11, 12). This mAb is often used to characterize resident macrophages although it also recognizes monocytes. In the rat a protein analogue of the F4/80 antigen is found (13). Previously we described the mAb ED2, recognizing a membrane antigen present predominantly on fixed tissue macrophages of rats (14). In different organs, ED2 positive cells with extensive cell processes are present which seem to be strongly fixed into the connective tissue. The bone marrow macrophages in erythropoietic clusters, the thymic cortical macrophages, liver Kupffer cells, and splenic red-pulp macrophages are all ED2 positive (14). Monocytes, dendritic cells, lymphocytes and granulocytes are negative for ED2. No cell types other than those belonging to the mononuclear phagocyte system are positive for ED2 (14). As detected by simultaneous demonstration of endogenous peroxidase activity on the ultrastructural level, ED2 expression was correlated with resident macrophages (15). In the present study, the in vitro expression, function, and structure of the antigen recognized by the mAb ED2 were examined. We examined the ED2 expression in several culture systems and in particular, the influence of reticular components, extra cellular matrix proteins (ECM) as well as reticular cells. We show that macrophage differentiation is enhanced under adhering, non proliferating conditions as well as in long-term bone marrow cultures (L TBC).
Materials and Methods Animals
Wistar rats were obtained from HSD/CPB, Zeist, The Netherlands, and kept under routine laboratory conditions.
90 . ELLIS BARBE, JAN G. M. C. DAMOISEAUX, ED A. Dopp, and CHRISTINE D. DI]KSTRA Bone marrow culture Bone marrow was obtained from young adult rats. The femora were excised aseptically, the end of the bones were removed, and the marrow was flushed out, using 5 ml of RPMI-1640 medium (Gibco, Uxbridge, U.K.) expelled from a 10 ml syringe through a 20-gauge needle. The released cells were washed by centrifugation and resuspended in culture medium consisting of: RPMI-1640 medium, 2 mM glutamin, 100 Ulml penicillin, and 100 glml streptomycin, supplemented with 20 % fetal calf serum (FCS) (Hyclone Laboratories Inc., UT, USA). The macrophage precursor cells were enriched by culturing the total bone marrow fraction for 3 days in 6 plastic culture flasks of 75 cm2 (each flask contained approximately 7.5 x 106 cells in 15 ml culture medium). The culture medium was supplemented with 20 % L929 cell conditioned medium (LCM) as a source of M-CSF (16). The non-adherent cell fraction containing the macrophage precursor cells, was harvested after 3 days. The in vitro expression of ED2 was studied by culturing macrophage precursor cells under different culture conditions. Non-adherent and adherent culture conditions were tested by culturing the macrophage precursor cells in teflon bags (17) or on covers lips placed in 24-well culture trays (16). The culture medium was differentially supplemented with 20 % LCM. The precursor cells were cultured in a concentration of approximately 106 cells/ml culture medium. Several different adherent culture conditions were tested by culturing the precursor cells on covers lips coated with poly-L-lysin (10 f!g/well) (Sigma, St. Louis, MO, USA», fibronectin (FN) (5 f!g/well) (Boehringer, Mannheim, Germany), or ECM. The ECM was produced by cells of a rat LTBC (see below). The adherent layer of a LTBC is capable of producing soluble factors which influence haemopoietic proliferation and/or differentiation and contain a variety of adhesion factors such as fibronectin, laminin, and type IV collagen (18). The supernatant of the LTBC was used for coating. For studying the ED2 positive macrophage population in bone marrow stroma, L TBC were used (18). Total bone marrow was cultured on covers lips placed in a 24-well tissue culture tray. After centrifugation, the total bone marrow fraction was resuspended in 75 ml culture medium with addition of 10 % newbornlfetal calf serum (NFCS) instead of FCS, divided over 3 culture trays containing coverslips, and cultured for several weeks. All experiments were done under optimal culture conditions at 37°C and 95 % air/5 % CO 2 in a humidified incubator. After culturing, cells were harvested from teflon bags and cytocentrifuged onto glass-slides. Glass-slides and coverslips were differentially stained for ED1, ED2 and acid phosphatase. Differential cell counts (100 cells) were performed on each preparation. Cells of the mononuclear phagocyte lineage were characterized by morphology, acid phosphatase activity and ED1 staining. All experiments were repeated at least three times. fmm unohistochemistry The following mAbs were used: ED1, a highly specific marker for cells of the mononuclear phagocyte lineage (14); ED2, a marker for a subpopulation of macrophages (14). As conjugate a rabbit-anti-mouse IgG/peroxidase (DAKOpatts, Copenhagen, Denmark) was used. Coverslips were rinsed in saline with 0.01 M phosphate buffered saline (PBS) pH 7.4 whereas cytospins were allowed to dry overnight. Coverslips and cytospins were fixed in aceton containing 2.5 % formol. After fixation the samples were rinsed with PBS and incubated with the mAbs. MAbs were diluted in PBS with 2.5 % bovine serum albumine (BSA) and used in optimal concentrations. After incubation with the first antibody for 30 min, the samples were rinsed thoroughly in PBS, incubated in conjugate for 30 min, washed again thoroughly in PBS, and stained for peroxidase activity with 0.5 mg/ml 3,3' -diaminobenzidinetetrahydrochloride (Sigma) in 0.05 M Tris-HCI buffer, pH 7.6 containing 0.01 % H 2 0 2 for 10 min. After rinsing in PBS the samples were slightly counterstained with haematoxylin, dehydrated and mounted in Entellan (Merck, Darmstadt, Germany). Control samples were incubated in PBS-BSA in the first step, with conjugate in the second step and examined for non-specific staining. Acid phosphatase activity was demonstrated according to the method of BURSTONE (19).
The ED2 macrophage differentiation antigen . 91 Radiolabeling and immunoprecipitation Peritoneal cells were surface labeled with Na l2S I (Amersham International, Amersham, U.K.) using lactoperoxidase (Sigma) and H 2 0 2 (20). Radiolabeled cells were lysed with 1 % Nonidet P-40 in 0.01 M triethanolamine-HCl, pH 7.8, 0.15M NaCI. Proteolysis inhibitors used are 1 mM phenyl methyl sulfonyl fluoride, 1 fAg/mlleupeptin, 1 fAg/ml pepstatin, 1 fAg/ml antipain. Nuclear debris was removed from the lysates by centrifugation at 15,000 x g for 5 min at 4°C. Preclearing of the lysates was done with normal mouse serum for 30 min succeeded by protein G Sepharose (Pharmacia LKB, Uppsala, Sweden) for another 30 min. Precleared lysates were incubated for 1 h with the ED2 or control mAb, succeeded by protein G Sepharose for 30 min. Immunoprecipitates were removed from the Iysates by centrifugation at 15,000 x g and were washed four times in 0.05 M Tris-HCl, pH 7.5, 0.5 % NP40, 5 mM EDTA, O.l5M NaCI. Electrophoresis and autoradiography SDS polyacrylamide gel electrophoresis was carried out on 10% acrylamide vertical slab gels according to LAEMMLI (20). Immunoprecipitates were dissolved by boiling for 5 min in electrophoresis sample buffer. The gels were run under reducing conditions, fixed, dried, and autoradiography was performed at -70°C using Kodak XAR-5 film. Adhesion assay To examine the adhesive capabilities of the ED2 antigen the adhesion assay modified after ROSEN et al. (21) was performed. Peritoneal cells were suspended in RPMI-1640 medium containing 1 % BSA (Organon Technika, Boxtel, The Netherlands), and plated at a density of 1 x 105 or 2 x 105 cells/well in 96-well plates of bacterial plastic (BP) (Greiner, Alphen aan de Rijn, The Netherlands). The plates were differentially coated with FN, ECM, or poly-L-Iysine as described above. After incubation for 30 min at 3rC, plates were washed three times in PBS and adherent cells were fixed with methanol. After staining with 0.2 % Toluidine Blue solution for 10 min, plates were washed in water, dried and the retained dye was solubilized in methanol. Stain was quantified by measuring absorbance at 620 nm in an automatic plate reader (Titertek Multiscan MCC/340). In antibody inhibition experiments, peritoneal cells were incubated with 10 fAl ascites of the mAb during 15 min at 4°C. Antibody excess was removed by extensive washing prior to the adhesion assay.
Results
The influence of adherent conditions on ED2 expression in bone marrow cultures To study the ED2 expression during adherent and non-adherent conditions, macrophage precursor cells were cultured on coverslips and in teflon bags. About 4 x 10 7 cells, containing macrophage precursors, could be h~rvested after culturing the total bone marrow on culture plastic for 3 days. Approximately 50 % of the harvested non-adherent fraction stained positive for EDl and acid phosphatase. About 3 % of the cells stained positive for ED2. When the harvested cells were cultured onto glass coverslips, almost all cells adhered after 3 days. This indicates that there was no selection by differences in adhesion. The percentage of mononuclear phagocytes increased from 50 % on day o via 80 % on day 7, 90 % on day 10 to almost 100 % in course of two
92 . ELLIS BARBE, JAN G. M. C. DAMOISEAUX, ED A. Dopp, and CHRISTINE D. DI]KSTRA
Days 01 culturing
Figure 1. Influence of adherent conditions on ED2 expression in bone marrow cultures. Bone marrow cells were cultured on glass-coverslips and in teflon bags. The culture medium was supplemented with 20 % FCS. The data represent mean percentages ± SD of macrophages positive for ED2.
weeks in both teflon and coverslip cultures. The percentage of macrophages positive for ED2 on coverslips increased from 3 to 90 % in course of one week and decreased thereafter to 25 % on day 14 of culturing (Fig. 1). There was however a variation in the level of ED2 expression observed. During culturing a maximum of 75 % of the cells stained weakly for ED2, whereas a maximum of only 15 % stained strongly for ED2. 100
% ED2 80
D ~
gil. weak 51
gil. strong st
60
40
20
0
Figure 2. Influence of LCM on ED2 expression in bone marrow cultures. Bone marrow cells were cultured on coverslips and in teflon bags. The culture medium was supplemented with 20 % FCS and 20 % LCM. The data represent mean percentages ± SD of macrophages positive for ED2.
The ED2 macrophage differentiation antigen . 93
In teflon, no increase of ED2 expression could be noticed. The ED2 positive macrophages remained between 3 % and 6 % during 14 days of culturing (Fig. 1).
The influence of LeM on ED2 expression Macrophage precursor cells were cultured in culture medium supplemented with 20 % LCM, on coverslips and in teflon bags. After 3 days of culturing in teflon and coverslip cultures, 80 % of the cells became positive for EDl and stained positive for acid phosphatase. On day 7, almost all cells were macrophages. On coverslips the percentage macrophages positive for ED2 increased from 3 to 62 % in course of 14 days, whereas the percentage of ED2 positive macrophages from the teflon culture, remained at a low level during culture time (Fig. 2). Addition of LCM to the culture medium stimulated macrophage proliferation, resulting in a high number of macrophage precursor cells. Culturing in the presence of LCM again resulted in a variable level of ED2 expression (Fig. 2).
ED2 expression in the presence of stromal components In different organs, adherent resident macrophages with extensive cell processes can be found which seem to be strongly fixed into connective tissue. To study the ED2 expression in relation to adherence to stromal components, bone marrow precursor cells were cultured on coverslips coated with several materials. Coating coverslips with FN or ECM stimulated the proliferation resulting in a higher number of macrophages than the number of macrophages on uncoated coverslips. Compared to glass, the
100
%
ED2
0
80
glas weak 5t
glas strong 51
EeM
F N
60
poly-L-Iysln
40
20
0
Days 01 cultUring
Figure 3. ED2 expression in a bone marrow culture in the presence of several adhesion components. Macrophages were cultured on coverslips differentially coated with FN, ECM, or poly-L-Iysin. The data represent mean percentages ± SD of macrophages positive for ED2.
94 . ELLIS BARBE, JAN G. M. C. DAMOISEAUX, ED A. Dopp, and CHRISTINE D. DI]KSTRA
presence of poly-L-lysin, FN, or ECM did not result in an extensive increase of ED2 expression (Fig. 3). To determine macrophage differentiation in LTBC, freshly isolated total bone marrow was cultured on coverslips. The culture medium was supplemented with 10 % NFCS. After 14 days of culture, a stromal monolayer was produced. Approximately 70 % of the cells stained positive for EDl and acid phosphatase and approximately 35 % of the monolayer cells stained positive for ED2.
Heterogeneity of the cultured cells The macrophages cultured in the presence of LCM showed a round morphology with a round oval excentric nucleus and a large cytoplasm fraction (Fig. 4a), whereas macrophages cultured in the absence of LCM had extensive cell processes with less cytoplasm (Fig. 4b). The cells that stained positive for ED2 also had predominantly extensive cell processes (Fig. 4c). On cytospins of the macrophages from the teflon bags, different
•••• B
! c ·
, 0
Figure 4. Morphology of macrophages cultured on coverslips. A: macrophages cultured for 3 days in the presence of 20 % LCM, stained for EDl, magnification 400x. B: macrophages cultured for 3 days in the absence of LCM, stained for EDl, magnification lOOOx. C: macrophages cultured for 7 days in absence of LCM, stained for ED2, magnification lOOOx. D: LTBC of 3 weeks, stained for ED2, shows a mixture of ED2 positive macrophages with extensive processes and ED2 negative fibroblastic cells, magnification 400x.
The ED2 macrophage differentiation antigen . 95
1
200
2
~
116 ~ 96~
68~
Figure 5. Analysis by immunoprecipitation of the antigens recognized by ED2. Resident peritoneal cells were surface labeled with 125 1, solubilized in detergent and immunoprecipitated with an antibody and Protein G Sepharose. 1mmunoprecipitates were subjected to SDS 10% PAGE under reducing conditions followed by autoradiography. Lane 1: ED2. Lane 2: irrelevant antibody control. The position of the molecular weight markers is given on the left side of the figure.
morphology could not be noticed in presence or absence of LCM. Except in morphology, a clear distinction could be made under all circumstances in the extent of ED2 expression. There was no gradual transition in expression, but only weak or strong expression, or no expression at all. In LTBC (Fig. 4d) the cells which did not stain positive for EDl had a fibroblastic morphology with a large oval nucleus with several conspicious nucleoli and a large cytoplasm fraction. The cells which stained positive for ED2 in the LTBC could be divided into two groups. One group seemed to adhere strongly to the glass, had extensive cell processes, made contact with surrounding cells and was part of the monolayer. The other group had a round appearance and adhered slightly to the monolayer or not at all. The cells which stained negative for ED2 were either fibroblastic cells or small round macrophages with a small round nucleus and a small cytoplasm fraction.
Molecular weight and adhesion assay The results of immunoprecipitation using the ED2 mAb and the Na 125 I_ labeled peritoneal macrophage lysates, showed that the ED2 mAb
96 . ELLIS BARBE, JAN G. M. C. DAMOISEAUX, ED A. Dopp, and CHRISTINE D. DIjKSTRA
immunoprecipitated three protein chains of Mr 175,000, 160,000, and 95,000 under reducing conditions (Fig. 5). This molecular weight has characteristics of the molecular weights of the integrins, a family of adhesive receptors (22). In order to test whether the ED2 antigen is a cell surface protein with adhesive properties, we studied the adhesion of peritoneal cells, which contain 70 % mononuclear phagocytes and approximately 30 % ED2 positive cells (14). The ED2 mAb was not able to inhibit macrophage adhesion (Table 1). To investigate the role of several adhering substrates, the cells were differentially coated with FN, ECM, or poly-L-Iysin. Coating with ECM resulted in a significant 80 % decrease in adherence compared to plastic. Poly-L-Iysin and FN coating resulted in a 50 % increase in adherence. Incubation of the peritoneal cells with the ED2 mAb did not result in adherence inhibition to these substrates (Table 1). Discussion The use of liquid bone marrow cultures offers an in vitro approach for studying macrophage differentiation (16, 18,23). The ED2 mAb recognizes a differentiation antigen on resident macrophages and is not expressed on monocytes and recently recruited inflammatory macrophages (14, 15,24). In freshly isolated bone marrow, almost 100 % of the typical mature macrophages is ED2 positive, whereas precursors are ED2 negative (23). In earlier studies, the expression of ED2 could not be induced under different non adherent culture conditions. In this study however, the ED2 antigen was inducible on bone marrow derived macrophages if the culture system provided the appropriate conditions for differentiation. The ED2 expression appeared to be restricted to environments where adhesive components are available. In vitro, no ED2 positive cells were observed when teflon bags were used for culturing the precursor cells. When precursors were cultured on materials to which mature macrophages could adhere, glass, FN, ECM, or polyL-lysin, ED2 was highly inducible. When LCM is added to cultures, Table 1. Adhesion assay
Bacterial Plastic Poly-L-lysin Extra-Cellular Matrix Fibronectin
Control
Control mAb
ED2 mAb
241 367 SO 363
215 ± 291 ± O± 261 ±
228 321 SO 313
± ± ± ±
10 20 30 10
10 10 0 20
± ± ± ±
10 10 10 60
Adhesion of peritoneal macrophages to plastic differentially coated with Poly-L-lysin, ECM, or Fibronectin. About 105 cells per well were plated in 96-well plates of bacterial plastic. Results are presented as mean absorbance at 620 nm ± SD (x 103 ) of triplicate experiments.
The ED2 macrophage differentiation antigen . 97
macrophage proliferation is stimulated, resulting in a higher number of mononuclear phagocytes but a relatively lower number of mature macrophages (16). This explains the fact that when LCM is added to the culture a lower number ED2 positive macrophages is observed. Several in vivo studies support the idea that adhesion is necessary to induce ED2 expression. After macrophage elimination in spleen using liposome-encapsulated dichloromethylene diphosphonate, it takes about 8 days before the red-pulp is repopulated by ED1 positive macrophages. Only after another week do these macrophages start to express ED2 (25). Also in ontogenetic development of the lymphoid organs the ED2 expression is observed about 1 to 2 weeks after the first macrophages have populated these tissues (26, 27). Apparently the ED1 positive macrophages require about a week to become ED2 positive after entry into a tissue, and thus need adherence to connective tissue. However, we showed that the ED2 antigen is also expressed on non-adherent cells in LTBC, that is after contact with the bone marrow fibroblastic cells. Also in vivo about 50 % of the free resident peritoneal macrophages express ED2 (14, 24). This indicates that interaction with adhesive components is necessary to induce ED2, but not for maintenance of the antigen expression. Macrophage adhesion plays an important role in a variety of processes including phagocytosis, inflammation, immunity and arteriosclerosis (21). Selective interaction with adhesive components, like FN, laminin, and vitronectin depends on specific plasma membrane receptors (28-30). ECM, consisting of a variety of proteins including FN, laminin, type IV collagen, and several growth factors, plays an important role in in vitro proliferation and differentiation of haemopoietic cells (31). Our study shows that macrophages exhibit differential affinity for these substrates as studied in the adhesion assay. They have high affinity for FN and poly-L-Iysin, but low affinity for ECM, although this mixture also contains FN. As stated above, many different substrate-specific plasma membrane receptors might be responsible for this differential affinity. In spite of this difference in affinity, we showed in the culture system that after three days nearly all cells adhered to the same extend to the four substrates used. Furthermore, the presence of poly-L-Iysin, FN, or ECM did not result in a difference in the percentage ED2 positive macrophages. However, when we take into account the higher proliferation rate on FN and in the presence of LCM, these two substrates might accelerate the induction of ED2 expression and therefore differentiation. The molecular weight of the ED2 antigen is suggestive for being a member of the integrin superfamily of adhesion molecules. Especially the 95 kDa subunit points in the direction of the LFA-l family, which is specific for leukocytes (22). However, the tissue distribution of the ED2 antigen is not in agreement with any of the members of the LFA-family (14, 22). Furthermore, we showed that the mAb ED2 was not able to inhibit adhesion.
98 . ELLIS BARBf:, JAN G. M. C. DAMOISEAUX, ED A. Dopp, and CHRISTINE D. DIJKSTRA
Taken together, our results show that ED2 is a very usefull tool for in vitro labeling of resident macrophages in rat. The in vivo functional relevance of the ED2 antigen needs further investigation.
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The ED2 macrophage differentiation antigen . 99 20. LAEMMLI, U. K. 1970. Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature 227: 680. 21. ROSEN, H., and S. GORDON. 1987. Monoclonal antibody to the murine type 3 complement receptor inhibits adhesion of myelomonocytic cells in vitro and inflammatory cell recruitment in vivo. J. Exp. Med. 166: 1685. 22. HYNES, R. O. 1987. Integrins: a family of cell surface receptors. Cell 48: 549. 23. DA~10ISEAUX, J. G. M. c., E. A. Dopp, R. H. J. BEELEN, and C. D. DIjKSTRA. 1989. Rat bone marrow and monocyte cultures: influence of culture time and lymphokines on the expression of macrophage differentiation antigens. J. Leukocyte BioI. 46: 246. 24. DAMOISEAUX, J. G. M. c., E. A. Dopp, J. J. NEEFjEs, R. H. J. BEELEN, and C. D. DIJKSTRA. 1989. Heterogeneity of macrophages in the rat evidenced by variability in determinants: two new anti-rat macrophage antibodies against a heterodimer of 160 and 95 kd (CDllICDI8). J. Leukocyte BioI. 46: 556. 25. VAN ROOIJEN, N., N. KORS, M. VAN DER ENDE, and C. D. DIjKSTRA. 1990. Depletion and repopulation of macrophages in spleen and liver of rat after intravenous treatment with liposome-encapsulated dichloromethylene diphosphonate. Cell Tissue Res. 260: 215. 26. VAN REEs, E. P., E. A. Dopp, C. D. DIjKSTRA, and T. SMINIA. 1985. The postnatal development of cell populations in the rat popliteal lymph node. An immunohistochemical study. Cell. Tissue Res. 242: 391. 27. VAN REES, E. P., C. D. DIjKSTRA, M. B. VAN DER ENDE, E. M. lANSE, and T. SMINIA. 1988. The ontogenetic development of macrophage subpopulations and Ia positive non-lymphoid cells in gut associated lymphoid tissue of the rat. Immunology 63: 79. 28. IGNOTZ, R. A., J. HEINO, and J. MASSAGUE. 1989. Regulation of cell adhesion receptors by transforming growth factor-~. J. BioI. Chern. 264: 389. 29. HUARD, T. K., H. L. MALINOFF, and M. S. WICHA. 1986. Macrophages express a plasma membrane receptor for basement membrane laminin. Am. J. Pathol. 123: 365. 30. GARCIA-PARDO, A., O. C. FERREIRA, J. VALINSKY, and C. BIANCO. 1989. Fibronectin receptors of mononuclear phagocytes: binding characteristics and biochemical isolation. Exp. Cell Res. 181: 420. 31. CAMBEL, A., M. S. WICHA, and M. LONG. 1985. Extracellular matrix promotes the growth and differentiation of murine hematopoietic cells in vitro. J. Clin. Invest. 75: 2085. Dr. J. G. M. C. DAMOISEAUX, Department of Cell Biology, Division of Histology, Medical Faculty, Vrije Universiteit, Van de Boechorststraat 7, 1081 BT Amsterdam, The Netherlands
