Indomethacin modulates plasma membrane-associated properties of macrophages

Prostaglandins Leukotrienes and Medicine 8:

301-310, 1982

INDOMETBACIN MODULATES PLASMA MEMBRANE-ASSOCIATED PROPERTIES OF MACROPBAGES E. Raxin1'3, A. Globersonl and E. Skutelsky2. Departments of ICell Biology and 'Membrane Research, The Weizmann Institute of Science Rehovot 76100, Israeland 3Harvard Medical School, Boston, MA 02115, USA (Reprint requests to AG)

ABSTRACT Changes in the plasma membrane of macrophages treated with indomethacin were investigated. A significant decrease in the binding of IgG was observed with indomethacin-treated cells, as compared to the control group. Electron microscopic examination did not show any significant morphological differences between the indomethacin treated macrophages and the controls. It was found, however, that indomethacin treatment resulted in a drastic increase in the binding capactiy of the cell membrane to cationized ferritin. It is suggested that indomethacin may have a modulating effect on the macrophage plasma membrane.

INTRODUCTION Prostaglandins (PGs) have been shown to modulate the level of tosis by macrophages. Hence, high concentrations of PGs (>lO inhibited (l-3), whereas low concentrations (lo-* mg/ml-10-4 mg/ml) enhanced the process of engulfment of sheep red cells (SRC) by mouse macrophages (3). Furthermore, indomethacin, an inhibitor of PG synthesis, suppressed phagocytosis (4-6). A clue to the mechanism by which macrophage function is regulated by PGs may be derived from changes occurring at the level of the.cell plasma membrane. Plasma membranes of macrophages and cell lines derived from macrophages contain an Fc receptor that binds antigen-antibody complexes and aggregated mouse IgG (7-9). In addition, surface-negative charges interfere with the recognition mechanism between macrophages and expelled erythroid nuclei (10) or old erythrocytes (11). In the present communication we show that macrophages treated with indomethacin have a decreased capacity to bind IgG and an increased capacity to bind cationized ferritin (CF). Furthermore, we show that PGE2 and PGF2, reverses the effects of indomethacin.

301

MATERIALS AND METHODS

Animals. C3H/DiSn male mice were obtained from the Animal Breeding Center of the Weizmann Institute of Science. Two- to 4-month old mice were employed throughout this study. Pharmacologic agents. Prostaglandins (PGs) and PGF2o (kindly donated by Dr. J. Pike of the Upjohn Co., Kalamazoo, MI) were dissolved in ethanol and then diluted in phosphate buffered saline (BPS), so that the final concentration of ethanol did not exceed 0.1% (v/v). Indomethacin (I-P-chlorobenzoyl-5methoxy--2-methylindol-3-acetic acid; Assia Chemical Laboratories, Tel Aviv) was dissolved in ethanol and prepared in the same fashion as the PGs. Preparation of 'lCr-labeled IgG-sensitized sheep red cells (IgG-SRC). SRC 10% (v/v) in PBS were sensitized b rabbit antiserum to SRC and labeled with 100 uCi sodium chromate (z ICr) solution (Radiochemical Centre, Amersham, UK), as recommended by the manufacturer. Macrophage preparation and treatment. Peritoneal macrophages were collected 4 days after an intraperitoneal (i.p.) injection of 4 ml thioglycollate (3%). The cells were suspended in 10 ml RPM1 medium in the presence or absence of indomethacin (1 ug/ml). PGE2 and/or PGFz,, each at the final concentration of 10 ng/m& wer'eadded to cultures to ether with indomethacin. The cells were washed and incubated (10 x 10i cells) in g-cm diameter untreated plastic dishes (Nunc A/S, Denmark) Incubation was carried out at 370C in a 5% CO2/95% air humidified atmosphere. After 3 h of incubation, the dishes were rinsed with PBS to remove the nonadherent cells and the cells were reincubated overnight in the same solutions. Assay of the Fc receptor. The binding of the IgG to the cells was evaluated as a measure of Fc receptors, by incubating the macrophages with 51Cr-IgG-SRC at 4OC for 30 min (12). Labeling of macrophages with cationized ferritin. Cationized ferritin (CF; 13) was purchased from Miles-Yeda (Rehovot, Israel). Macrophages which were detached from the plastic petri dishes were fixed with Karnovsky's fixative (14) and labeled with the CF (15). Processing for electron microscopy. All fixed cells in the petri dishes were washed x6 with Verona1 acetate buffered saline (VBS) and refixed in 1% OS04 in VBS for 1 h. Each sample was placed in a glass test tube and dehydrated in graded ethanol solutions, stained with saturated aqueous uranyl acetate, and embedded in Epon (16). Thin sections were obtained with an MT-2 Sorval microtome, mounted on uncaoted 400 mesh copper grids and coated with carbon. A Joel-100 A electron microscope was used at 100 kV. Analysis of labeled charge density on cell surface. Ferritin particles were counted on prints (100,OOOxfinal magnification) on tangential sections of macrophage cell membranes with 5 to 10 areas of 0.01 pm2 taken at random from 10 to 20 different macrophages. 302

PGE2 radioimnunoassay. Culture fluids were assayed for PGEi by radioimmunoassay (17).

RESULTS The kinetics of the effect of indomethacinonIgG binding were studied by measuring cell-bound " Cr-IgG-SRC after varying times of exposure of cells to the drug (Fig. 1). A significant reduction in the binding was observed by 5 h of exposure and maximum inhibition (50%) occurred by 12 h We then examined the effect of various concentrations of indomethacin on both PGE2 release and binding of IgG after 12 h of treatment (Table 1). When the cells were treated with indomethacin at 1 pg/ml there was a parallel decrease in PGE2 and IgG binding.

t 0

I

I

I

5 IO Time of Treatment (hrz

I

20

FIGURE

1. Kinetics of the effect of indomethacin on the binding of 'ICrIgG-SRC with peritoneal macrophages.

Ten x 106 macrophages obtained from the peritoneal cavities of C3H/DiSn male mice 4 days after i.p. injection of thioglycollate were exposed for varying times to indomethacin (1 ug/ml). SRC were sensitized by rabbit antiserum and labeled with 100 $i chromium-51 solution. The bindin of IgG to the cells was measured after incubating the macrophages with @'Cr IgG for 30 min. The pH of the medium remained unchanged by indomethacin treatment. Percent inhibition of IgG binding = 100 minus (cpm experimental group divided by cpm control group) multiplied by 1OC. Each point on the graph represents the mean + standard error of three replicate samples.

303

TABLE 1.

Effect of various concentrations of indomethacin on PGE2 release and IgG binding by peritoneal macrophages

Indomethacin (pg/ml)

PGE2 (ng/106 cells)

0

10.4 9.9 9.7 5.9 0.5

0.01 0.05 0.1 1.0

+ 1 _+0.8 + 1.2 f 0.6 f 0.1

IgG binding (cpm) 4520 4532 4497 3376 2129

f k k * +

256 379 306 205 346

Ten x lo6 macrophages were exposed for 12 h to various concentrations of indomethacin. Culture fluids were assayed for PGE by radioimmunoassay. Each value represents the mean 2 standard error of three replicate samples.

We then examined the effect of indomethacin on PGE2 release, as a function of time. As shown in Table 2, there was a time-related decrease in PGE2 in the supernatants of cultures treated with 1 ug/ml indomethacin. This inhibition of PGE release was parallel to the inhibition in IgG binding (Fig. 1). The fact that the IgG binding was decreaed by 50% while the release of PGE was decreased by 98% suggests that PG is not the sole factor affecting the IgG binding.

TABLE 2.

Time (h)

2 5 8 12

Kinetics of PGE indomethacin

release by macrophages treated with

PGE:!hg/106 macrophages) + Indomethacin 2.5 1.9 0.6 0.05

in0.2 * 0.1 -I0.2 ii0.02

- Indomethacin 2.3 4.9 9.3 10.2

f + + +

0.4 0.3 0.3 1.1

Ten x lo6 macrophages were exposed to indomethacin, 1 ug/ml, for various periods of time. Culture fluids were assayed for PGE2. When the macrophages were exposed for 12 h to indomethacin alone and to indomethacin with PGE2, PGF2u or a mixture of both, the amount of 51CrIgG-SRC binding was the same as the control values (Fig. 2). The concentrations of PGs which reversed the indomethacin effect were the same as those found in the supernatants of 10 x lo6 macrophages incubated overnight at 37oC, in an atmosphere of 5% Cop, 95% air.

304

Control

Indometh.

i-l

1

I ndometh. +

PGE2 Indometh. +

’

PGF2a Indomcth. +

’

PGE2+PGF2a 6

I

I

I

I

I

I

0

I

2

3

4

5

6

cpm

x103

FIGURE 2. Effect of PGs on the binding of 51Cr-IgG-SRC to peritoneal macrophages. Ten x lo6 macrophages were exposed to indomethacin (1 pg/ml), with or without PGs (PGE2 and PGFz,, 10 ng/ml each) for 12 h. Controls were treated with medium only. Each point represents the mean + standard error of three replicate samples. We then evaluated the effect of indomethacin and PGs on the distribution of cell surface anionic sites by examining the distribution of CF particles attached to tangentially sectioned membrane areas on the individual macrophages. Indomethacin treatment markedly increased the binding of CF to cell membranes (Fig. 3), as compared to the untreated control cells. However, subsequent treatment of the cells with PGs (PGE2 and/or PGF2u) restored the binding capacity of the cell surface to CF to its original level. These results were quantitated by counting the CF particles on micrographs of tangentially sectioned membranes, as summarized in the histogram (Fig. 4). The same pattern of increase in CF binding to indomethacin treated cells was obtained when the macrophages were fixed by Karnovsky fixative before detachment from the dishes.

305

FIGURE 3. Electron micrographs of tangentially sectioned areas of surface membranes of macrophages labeled with cationized ferritin. (A) Untreated macrophages. Macrophages exposed methacin and (C) indomethacin + PGE2 for 23 h. those presented in (C) were obtained with cells tin + PGF2u or indomethacin + a mixture of PGE2

to (B) 1 ug/ml indoResults similar to treated with indomethaand PGFza.

DISCUSSION Prostaglandins at physiological concentrations were found to be essential mediators in the binding of IgG to macrophages and in the distribution of anionic sites on the cell surface. These results shed new light on our previous findings that PGs enhance the binding capacity of macrophage Fc and Con A receptors (5). Hence, the information presented here links the effect of absence of PGs on the expression of Fc receptors to the redistribution of membrane charges, a phenomenon which influences the In cell interactions, it is accessibility and/or affinity of receptors. commonly believed that the surface changes (which are mainly contributed

306

30 20 10 +

30

=

20

E IO w a 30 20 IO

CATIONIZED

FERITIN(

Particles

per 0.01pm2)

FIGURE 4. Histogram showing distribution of labeling with cationed ferritin of macrophage plasma membrane. (A) Control; (B) indomethacin, and (C) indomethacin + PGE2. Results similar to those presented in (C) were obtained with cells treated with indomethacin + PGF2o or indomethacin + a mixture of PGE2 and PGF2o. Ferritin particles were counted on prints (100,000x final magnification) on tangential sections of macrophage cell membranes with 5 to 10 areas of 0.01 pm2 taken at rar.domfrom 10 to 20 different macrophages.

by the sialic acid) have a general "protective" role expressed mainly by a "masking" vs. an "exposure" of cryptic recognition sites on the cell membranes. Upon exposure, the latter sites are in turn capable of interacting directly with other cell membranes and/or plasma antibodies (18-20). Thus, it would be anticipated that laterations in the normal level and distribution ofsurface sites would lead to changes in the binding of ligands to their respective receptors. Surface anionic sites have a direct effect on phagocytic activity. It has previously been shown that attachment and phagocytosis of negatively charged particles by human blood monocytes is enhanced after neuraminidase treatment (20). It has been suggested that this enhancement of phagocytosis is due to the reduction in the electrostatic repulsive forces between the phagocyte and the particles of the same electrostatic charge. In addition, membrane negative charges of cells seem to play a role in the recognition mechanism between macrophages and "old" RBC (11). "Old" RBC have a lower surface negative charge than "young" RBC (21-23) and it has been suggested that this reduced surface negative charge may facilitate the closer contact between the membranes of these "old" RBC and macrophages by decreasing intercellular repulsion forces (24). 307

Presumably similar considerations also apply to the surface charge properties of the macrophages. The inverse relationship between Fc receptors and anionic sites influenced by the indomethacin treatment, together with the finding (5) that PGs enhance the expression of macrophage Con A receptors, are in good agreement with the inverse relationship between levels of Con A receptors and anionic sites in.different leukoid and erythroid cell types (10). Although the precise mechanisms by which PGs modulate the expression of Fc receptors and the distribution of surface negative charges is still unkonwn, the effects of PGs on plasma membrane properties were reported previously in other experimental models. Kury et al. (25) showed that PGE2 and PGEl influenced the fluidity of red cell membranes. Work under way had indeed confirmed display changes in the rigidity of the plasma membrane of indomethacin-treated peritoneal macrophages (26). Accordingly, it could be suggested that change in fluidity may lead to modified motility of the macrophage cell membrane components in the absence of PG and thus to a possible rearrangement of IgG receptors and anionic sites. An interesting similarity exists between the density and labeling pattern of CF on the indomethacin-treated macrophages to that previously observed on macrophages derived from thioglycollate-stimulated newborn mice (15). In the latter case, despite the elevated surface charge density, phagocytosis of turkey RBC was enhanced, as compared to macrophages derived from adults. This may also support the assumption that the lack or "masking" of receptors sites, rather than elevation of surface charge density, is important in the modification of binding of these cells to IgG-SRC. The possibility that, similarly to the indomethacin-treated macrophages, incomplete capability of newborn cells to synthesize PGs may be associated with the high density of anionic sites on these cells will be a subject for further studies. Acknowledgement. The authors acknowledge the helpful advice of Dr. A. Rifkind, Cornell University Medical College.

REFERENCES 1.

Weissman, G., Dukor, 0. and Sessa, G.: Studies on lysosomes: Mechanisms of enzyme release from endocytic cells and a model for latency in vitro. In: Immunopathology of Inflammation (B.K. Forscher and J.C. Houck, eds.), Exceprta Medical Foundation, Publishers, Amsterdam, 1971, p. 107.

2.

Weissman, G., Zurier, R.B. and Hoffstein, S.: Leukocytic proteases and the immunologic release of lysosomal enzymes. Am. J. Pathol. -68: 539, 1972.

3.

Razin, E., Bauminger, S. and Globerson, A.: Effect of prostaglandins on phagocytosis of sheep erythrocytes by mouse peritoneal macrophages. J. Reticuloendothel. Sot. 23: 237, 1978.

308

4.

Brune, K., Glatt, M., Kalin, II.,and Peskar, B.A. Pharmacological control of prostaglandin and thromboxane release from macrophages. Nature 274: 261, 1978. --

5.

Razin, E. and Globerson, A. The effect of various prostaglandins on plasma membrane receptors and function of mouse macrophages. In: "Function and Structure of the Immune System" (W. Muller-Ruchholz and H.K. Muller-Hermelink, eds.),Plenum Publishing Corp., New York, 1979, p. 415.

6.

Razin, E., Zor, U. and Globerson, A. Function of macrophage prostaglandins in the process of phagocytosis. In: Adv. Exp. Med. Biol. 121A: 413, 1980. --

7.

Heusser, C.H., Anderson, C.L. and Grey, H.M. Receptors for IgG: Subclass specificity of receptors on different mouse cell types and the definition of two distinct receptors on macrophage cell line. J. Exp. Med. 145: 1316, 1977.

8.

Unkeless, J.C. The presence of two Fc receptors on mouse macrophages: Evidence from a variant cell line and differential trypsin sensitivity. J. Exp. Med. 145: 931, 1977.

9.

Walker, W.S. Separate Fc receptors for immunoglobulins IgG2a and IgG2b on an established cell line of mouse macrophages. J. Immunol. 116: 911, 1976.

10.

11.

Skutelsky, E. and Farquhar, M.G. Variations in distribution of Con A receptor sites and anionic groups during red blood cell differentiation in the rat. J. Cell Biol. -71: 218, 1976. Danon, D., Marikovsky, Y. and Skutelsky, E. The sequestration of old red cells and extruded erythroid nuclei. in: "Red Cell Structure and Metabolism" (B. Ramot, ea.), Academic Press, New YorkLondon, 1971, p. 13.

12.

Atkinson, Y.P. and Parker, C.W. Modulation of macrophage Fc receptor function by cytochalasin-sensitive structures. Cell. Immunol. 33: 353, 1977 -

13.

Danon, D., Goldstein, L., Marikovsky, Y. and Skutelsky, E. Use of cationized ferritin as a label of negative charges of cell surfaces. J. Ultrastruct. Res. -38: 500, 1972.

14.

Karnovsky, M.J. A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. Cell. Biol. -27: 137a, 1965.

15.

Hardy, B., Skutelsky, E., Globerson, A. and Danon, D. Ultrastructure differences between macrophages of newborn and adult mice. J. Reticuloendothel. Sot. -19: 291, 1976.

16.

Luft, J.H. Improvement in epoxy resin embedding methods. phys. Biochem. Cytol. 2: 409, 1961.

17.

Bauminger, S., Zor, U. and Lindner, H.R. Radioimmunological assay of prostaglandin synthetase activity. Prostaglandins 4: 313, 1973.

18.

Lloyd, C.W. Sialic acid and the social behavior of cells. w. Rev. 50: 325, 1975. --

309

J. Bio-

19.

Skutelsky, E., Marikovsky, Y. and Danon, D. Immunoferritin analysis of membrane antigen density. A. Young and old human red blood cells. B. Developing erythroid cells and extruded erythroid nuclei. Eur. J. Immunol. -4: 512, 1974.

20.

Weiss, C. Neuraminidase sialic acids and cell interactions. J. Natl. Cancer Inst. -50: 3, 1973.

21.

Danon, D. and Marikovsky, Y. Difference de charge electrique de surface entre erythrocytes jeunes et ages. Comptes Rendus Acad. Sci., Paris 253: 1271, 1961.

22.

Marikovsky, Y. and Danon, D. Electron microscope analysis of young and old red blood cells stained with colloidal iron for surface charge evaluation. J. Cell. Biol. -43: 1, 1969.

23.

Skutelsky, E. and Danon, D. Reduction in surface charge as an explanation of the recognition by macrophagesof nuclei expelled from normoblasts. J. Cell. Biol. -43: 8, 1969.

24.

Danon, D. Biophysical aspects of red cell aging. In: "Physiology and Pathology of Human Ageing," Academic Press, New York-San Francisco-London, 1975, p. 95.

25.

Kury, P.G., Ramwell, P.W. and McConnell, H.M. The effect of prostaglandin El and E2 on the human erythrocytes as monitored by spin labels. Biochem. Biophys. Res. Commun. -56: 478, 1974.

26.

Razin, E., Rivnay, B. and Globerson, A. Prostaglandins as modulators of macrpphage development from bone marrow. J. Reticuloendothel. Sot. 30: 239, 1981.

310