Immunol. Cell Biol. (1990)67. 137-142

A serum factor for macrophage activation after in vitro dodecylglycerol treatment of mouse lymphocytes Sadamu Homma't, Irving Millman^ and Nobuto Yamamoto'* 'Department of Microbiology and Immunohgy. Hahnemann University School of Medicine. Philadelphia. PA I9I02. and -Fox Chase Cancer Center. 7701 Burholme Ave, Philadelphia. PA 19111. USA (Submitted 25 August 1989. Accepted for publication 5 February 1990) Summary Alkylglyccrols, inflammation products of cancerous tissues, are potent macrophage activatingagents. Abriefffi I'/fro treatment (30 min) of mouse peritoneal tells with a low concentration (50 ng/mL) of dodecylglycerol (DDG) in 10% foeial calf scrum supplemented RPMI-1640 medium (FCS medium) activates macrophagcs for Fc-rcccptor mediated ingcstion activity, A serum ractor(s) was shown to be required for the activation ofmacrophages.Whcn non-adherent cells were treated with rac-sn-l(.l)-dodccylglyccrol (DDG) in a serum frec-01% egg albumin supplemented RPMl medium (EA medium) for 30 min and cultured in FCS medium for 2 h. the resultant conditioned medium contained a signal factor able to activate macrophages (macrophage aciivaiing factor). A conditioned medium prepared wiih electrophoresed serum a;-glabulin fraction in EA medium markedly enhanced activation ol" macrophagcs. Incubation of DDG-trcated non-adherent cell ghosts in EA medium containing o;-globulin also produced the macrophage activating signal factor. Therefore, it is concluded that a serum factor in a:-globulin fraciion is processed by pre-existing functions or enzymes of DDG-trealed non-adherent cell membrane lo yield a macrophage aetivaling signal factor.

Fc-receptor but not C3b receptor (1.2.6,7). In vitro treatment of macrophages alone with lysophosphatidylcholine (lyso-Pc) or with rac-snl(3)-dodeeylglycerol (DDG) results in no enhaneed ingestion aetivity (1.2,7). However, cultivation of a mixture of adherent (macrophage) and non-adherent (B and T) cells with lyso-Pc. other lysophospholipids or DDG for 3 h produces a markedly enhanced Fc-receptor mediated ingestion activity of maerophages. implyingsignal transmission from non-adherent cells to maerophage (1-3. 7). Incubation of untreated adherent eells with DDG-treated nonadherent eelis in a medium containing 10% foetal ealf serum supplemented RPMI-1640 (FCS Correspondence: Dr Nohuto Yamamolo. Departmedium) developed a greatly enhaneed ingesment of Microbiology and Immunology. Hahnemann tion activity of maerophages. When 0 1 % egg LJniversiiy Sehool of Medicine. Philadelphia. PA albumin (EA) was substituted for 10% foetal calf 19102. USA. serum (FCS) in medium for development of ^On leave from ihe 1st Department of Internal maerophage ingestion aetivity. a greatly reduced Medicine, The Jikei University School of Medicine, Tokyo. Japan. ingestion aetivity was observed. Therefore, we .Abbreviations used in this paper. BCG, Bacillus Cal- tested the hypothesis that a serum factor(s) plays mctlV'Gucrin: C. complement: DDG, rae-sn-l(3)a role in theaetivation of macrophages. We fracdodecylglyccrol; DBP, Vitamin Di binding protein: E, tionated serum hy electrophoresis and found sheep erylhrocytcs; EA, egg albumin: E.A medium. EA that a2-globulin is required for macrophage actisupplemented RPMI-1640 medium: FCS. foetal calf vation. In this communication we report a role serum: FCS medium; FCS supplemented RPMI-1640 of serum a^-globulin in activation of mouse perimedium: lyso-Pc, lysophosphalidylcholine: PBS, toneal macrophages. phosphatc-butfered saline. INTRODUCTION Administration of Bacillus Calmette-Guerin (BCG) and other bacterial agents to cancerous tissues induces innammation. Inflamed cancerous tissues produce hydrolysed lipids. alkyllysophospholipids and alkylglycerols as well as lysophospholipids (1-3), because cancerous eells contain alkylphospholipids and monoalkyldiacylglycerols (4.5). Administration of lysophospholipids or alkylglycerols to miee stimulates maerophages to ingest target cells via

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MATERIALS AND METHODS .4nimal.s Female BALB/c mice, 7-12 weeks of age, were obtained from the Jackson Laboratories. Bar Harbor, ME and were fed Purina Mouse Chow and water aii libitum. Chemicals and reagents Elher analogue of monoglyteride, rac-SN-U3)dodecylglycerol (DDG) was synthesized by the method of Baumann and Mangold (8) by Evan Gustow and R. A. Pieringer. Temple University. Potato starch was purchased from J. T. Baker Chemical Co. Phillipsburg, NJ. In vitro treatment of peritoneal cells Resident peritoneal eells were harvested from untreated mice, processed and quantified as described previously (1). After 30 min incubation at 37°C in a humidified 5% CO; Incubator to allow macrophage adherence on to the 12 mm coverglasses which had been placed in 1 6 mm lissue culture wells, these peritoneal cells (mixture of non-adherent and adherent cells) were incubated in FCS medium containing 50 ng DDG/mL. After 30 min incubation, non-adherent cells and residual DDG were removed and the adherent ceils were washed three limes in Dulbecco's phosphate buffered saline (PBS) and cultured in FCS medium for 3 h prior to ingestion assay of macrophages. Preparation of conditioned media of DDG-treated non-adherent cells Sinee a large number of non-adherent eells was necessary for the preparation of conditioned media of DDG-trcated or untreated non-adherent cells in media containing serum or various scrum fractions. splenic non-adherent cells were used. Spleen cells from BALB/c mice were collected and processed as deseribed by Tyan and Ness (9). The cells were suspended in FCS medium, placed in 100 mm x 15 mm plastic dishes and ineubated in a 5%CO2 incubator for 30 min to allow adherence of maerophages (adherent cells). Non-adherent eells were collected, washed in PBS and treated with 50 ng DDG/mL in serum free-0-1% egg albumin supplemented RPMI medium (F.A medium) for 30 min, washed in PBS and cultured in FCS medium or EA medium containing various serum fractions for 2 K. The cells were removed by low speed centrifugation (a table top centrifuge 1500 r/min, 10 min). The resultant conditioned media were tested for activation of macrophagesby 3 h cultivation wiih peritoneal adherent cells. Ingestion assay Ingestion of sheep erythrocytes (E)-coated with immunoglobulin G. ElgG. was determined as described by Bianco c/tj/. (10). In brief, washed E (Dutchland. Denver. PA) were coated with subaggiutinating dilutions of purified rabbit anti-E, IgG fraction (Cordis laboratories. Miami, FL). For the Fc-receptor-

mediated ingestion assay. 0 5 mL of a 0 5%suspetision ofthe above conjugates in RPMI-1640 medium without FCS was overlayed on each macrophage coated (monolayer) coverglass and incubated at 3 7 ^ in a humidified 5% CO2 incubator for I h. Non-inlernalized erythrocytes were lysed by immersing the coverglasses in a hypotonic solution ( 1 : 5 PBS) for 3-8 s. The macrophages were fixed wiih methanol. air dried and stained with Giemsa stain and ingestion was quantified microscopically. The data are expressed as the ingestion index according to Bianco et al. (10). Ingestion index = the percentage of macrophages which ingested erythrocytes X average number of erythrocytes ingested per macrophage. Preparation of splenic non-adherent cell ghosts Splenic noti-adherent eells were suspended in a hypotonic PBS (1:10 PBS) and allowed to stand for 30 min at room temperature. The resultant eell ghosts were washed twice by eentrifugation (3000 r/mins, 10 min) in PBS. Microscopic analysis revealed thai this procedure proves suecessful in preparing 100% nonadherent eell ghosts. Fractionation offoeial calf serum by preparative starch block electrophoresis Purified potato starch (453 5 g or I lb) (J. T. Baker. Phiilipsburg. NJ) was washed with 2 L ofO 05 mol/L barbital bufler. pH 86 (Sigma. St Louis. MO) over a Buchner funnel under vacuum. A slurry ofthe starch in the same buffer was poured into a plastic fratne (27-9x1 7-8 cm or 1 Ix 7 in)loadcpthofapproximately 19 em (0-75 in). The starch slurry was allowed to set and excess buffer was removed by blotting ihe surface with paper towels. A trough approximately I 5 2 cm (6 in) wide and 1-3 cm (V; in) in depth was cut into the block. oft~ centre, approximately half the distance to the edge ofthe frame. This trough was loaded with 4 mL of FCS coloured blue with a t raec amount of bromphenol blue. The starch block was ihen transferred to the eold room (5°C) and plaecd over luo 2 L beakers containing barbital buffer connected to the edges of the block with ihiek filter paper wicks: 600 V (5 mA) was applied overnight. When the blue colour of the albumin fraction had traversed the starch block close to the paper wick at the anode, the unit was disassembled and the starch block was cut into nine I 27 cm (Vi inch) sliecs. Eaeh sliee was transferred into a large centrifuge tube and eluted with Dulbecco's PBS (pH 7-3) overnight in the cold. Nine 8 mL PBS fractions were obtained. An aliquot (6 mL) of each fraction was concentraitd 2-fold using Amicon Centriprep 30" concentrators (Denver. MA). These fractions were submitted to bioassay for detection ofthe serum factor for macrophage activation. Immunoelectrophoresis of serum fractions Photographic glass plates (3 25 x 4 25 cm), precoated with 2% Difco Agar Noble (dried at 60°C overnight), were eovered with 1 5 mL of I • 1% agarose (Sea Kem ME. FMC Corp.. Rockland. ME) dissolved in barbiial buffer. 0 05 mol/L, pH 8-6. These plates were

SERUM FACTOR FOR MACROPHAGE ACTIVATION

139 i FA medtum I FCS medium

0

FCS

Fig. I. Immunoelectrophoresis oi' FCS fractions. Arrow points to a^-globulin of fraction no. 7. Lesser amounts of a2-globulin are seen in fraction nos 6 and 8. The last well contains FCS. All troughs except ihe one between fraction nos 6 and 7 werefilledwith goat antibovine serum. stored at 5'C prior to use. Aliquots (19 nL) of each serum fraction were electrophoresed for approximately I h at 80 V. 25 mA. Troughs were filled with goat anti-bovine serum and incubated at room temperature overnight. Plates, washed with PBS overnight, were dried and stained with Coomassie Blue (see Fig. 1).

RESULTS In vitro treatment of peritoneal cells with dodecylglycerol for activation of macrophages A brief treatment (30 min) of a mixture of peritoneal non-adherent and adherent eells with 50 ng DDG/mL in FCS medium results in a greatly enhanced ingestion of IgG-eoated target ceils but not lgM-coated target cells with complement (3). Since DDG treatment of adherent cells (macrophages) alone does not enhance ingestion activity of tnacrophages (3). we inferred that a signal factor(s) for macrophage activation is transmitted from non-adherent cells to adherent cells during the brief DDG treatment period. To identify such a signal factor, a serum-free medium is required lor cultivation of these cells. When peritoneal cells were treated with 50 ng DDG/mL of a serum free- 0 1 % EA medium for 30 min and cultured in EA medium for 3 h. a very small increase in ingestion activity of macrophages was observed (Fig. 2). In contrast. 30 min treatment ofthe peritoneal cells in FCS medium produced a greatly enhanced activation of macrophages as shown in Fig. 2. These obser-

100

200 Ingestion Index

300

400

Fig. 2. Enhancement of maerophage ingestion activity by treatment of peritoneal cells with DDG in either EA (stipple) or FCS (solid) medium. Mouse peritoneal cells were collected and incubated in either EA or FCS medium at 3 7 ^ for 30 min for adhesion of macrophages. Cells were treated with 50 ng DDG/mL in either EA or FCS medium for 30 min. After removal of non-adherent cells and residual DDG by washing in PBS. the adherent cells were cultured for 3 h at 37°C in a CO2 incubator in either EA or FCS medium prior to ingestion assay. Bars represent s.d.

vations suggest that a factor(s) contained in serutn is required for activation of macrophages. Transmission of signalling factor between non-adherent and adherent cells Non-adherent cells were treated with 50 ng DDG/mL in EA medium for 30 min. washed in PBS and cultured in FCS medium for 2 h. The resultant conditioned medium of the DDGtreated non-adherent cells was added to adherent cells and cultured for 3 h. A greatly enhanced ingestion activity of macrophages was observed as shown in Table I. However. 3 h cultivation of Table 1. Activation of macrophages by conditioned medium of DDG-treated non-adherent eells. Culture medium for adherent cells Fresh FCS mediurn Conditioned medium" Untreated DDG-treated

Ingestion Index 98 ±6

104±8 252 ± 14 *Mouse peritoneal cells were collected, placed in 24-well plates and incubated for 30 min for adherence of macrophages. Non-adhereni cells were separated from each well (3-5 X lO"* cells) and treated with 50 ng DDG/mL in EA medium for 30 min. After washing with PBS. ihe non-adherenl cells were cultured in 0.5 niL of FCS medium for 2 h at 37''C. The resultant conditioned medium were used as a medium for cultivation of adherent cells for macrophage ingestion assay.

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adherent cells with conditioned medium ofthe untreated non-adherent cells resulted in no enhanced ingestion activity of macrophages (Table 1). These results indicate that the DDGtreated non-adherent cells, if serum is present, generate a signal factor for macrophages to develop ingestion activity. Search for serum factor required for activation of macrophages Since a serum factor(s) is required for activation of macrophages, we searched for such a serum factor by electrophoretic fractionation. Foetal calf serum waselectrophoresed in a starch block (see methods). Each fraction was added to DDG-treated splenic non-adherent cells in EA medium and cultured for 2 h. The resultant conditioned medium was added to adherent cells for 3 h cultivation prior to ingestion assay. As shown in Fig. 3, cultivation of adherent cells with the conditioned medium prepared with fraction no. 7 of electrophoresed serum produced a greatly enhanced ingestion activity of macrophages. With fraction no. 6, a lesser degree of macrophage activation was observed. No other fraction had significant activity for macrophage activation. When analysed by

immunoelectrophoresis, fraction no, 7 (also nos 6 and 8) contained goat anti-bovine immunoreactable material, mainly in the a2-globulin region as shown in Fig. 1. Macrophage activating signal factor was produced by incubating DDG-treated nonadherent ceil ghosts in EA medium containing 02-globulin fraction When peritoneal cells (mixture of non-adherent and adherent cells) are treated with 50 ng DDG/mL in FCS medium for 30 min and washed to remove residual DDG and nonadherent cells, the adherent macrophages develop a greatly enhanced ingestion activity after 3 h cultivation (3). Thus, a rapid signal transfer from non-adherent cells to adherent cells must have occurred during a 30 min DDGtreatment period. Since a stepwise contribution of both non-adherent (B and T) cell types is required for development of macrophage activating signal (3), each non-adherent cell type should begin to process signals) in less than 15 tnin. This would suggest that a serum factor(s) Is rapidly modified by pre-existing, rather than inducible. functions or enzymes ofthe B and T cells. Therefore, we prepared enucleated cell ghosts of splenic non-adherent (B and T) cells by hypotonic shock in 1/10 PBS. The splenic non-

2S0 DDG treated

200

PDG

50

0 1

2

3

4 5 6 7 Fraclfon number

B

9

Fig, 3. Activation of macrophages usingcondilioncd medium of DDG-treated non-adherent cells prepared with EA medium containing electrophoretic fractions of FCS. FCS was fractionated by preparative siarch block electrophoresis as described in the methods. Mouse splenic non-adherent cells (I O''/mL) were treated with 50 ng DDG/mL in EA medium for 30 min. After washing, the 10^ cells were suspended in 0.5 mL of EA medium containing various fractions (nos 1-9) of FCS. placed in each well and incubaled for 2 h. The resultant conditioned medium was used as a medium for 3 h cultivation of untreated peritoneal adherent cells prior to macrophage ingestion assay. Bars represent s.d.

100

200

300

400

Ingcslion Index

Fig. 4. Macrophage activation using conditioned medium of DDG-treated non-adherent cell ghosts prepared in EA medium containing a^-globulin fraction of FCS, Mouse splenic non-adherent cell ghosts were prepared as described in the methods and treated with 50 ng DDG/mL in EA medium for 30 min al 37''C. After washing with PBS, DDG-treated non-adherent cell ghosts were incubated in EA medium containing a2-globulin fraction (fraction no. 7) for 2 h at 37°C. The resultant conditioned media were used as a medium for 3h cultivation of untreated peritoneal adherent celts for macrophage ingestion assay. A small dose of a2-globutin fraction (100-fold diluted sample of Fig. 3) was sufficient to activate macrophages. Bars represent s.d.

SERUM FACTOR FOR MACROPHAGE ACTIVATION

adherent cell ghosts were treated with 50 ng DDG/mL in EA medium for 30 min, washed in PBS and incubated in a2-globulin (electrophoresed fraction no. 7) supplemented EA medium for 2 h. After removal of the non-adherent cell ghosts, the medium was used for 3 h cultivation of peritoneal adherent cells. A greatly enhanced Fc-receptor mediated ingestion activity of macrophages was observed as shown in Fig. 4. Therefore, we concluded that a serum component of ai-globulin fraction is rapidly modified by pre-existing membranous functions or enzymes of B and T cells to yield macrophage activating factor. DISCUSSION The present paper demonstrates that a2-globulin contains a factor required during cultivation of DDG-treated peritoneal cells for activation of macrophages. Fraction no. 8 of a:-globulin shown in Fig. 1, however, had no activity for activation of macrophages. suggesting that one {not all) of the a2-globulin components is responsible for macrophage activation. One of the major components in serum a2-globulin is a Vitamin D3 binding protein (DBP). Abe et al. (11) reported that Vitamin Di sterol. 1.25dihydroxyvitamin Di (1.25(OH)2D.i). activates alveolar macrophages as measured by increa.se in Fc-receptor and induction of cytotoxicity. LJndifierentiated tumour haematopoietic cells such as myeioid leukaemia (12), promyelocytic leukaemia cell line (I 3) and a lymphomacell line (14) can also be induced by 01-10 |imol/L l.25(OH)2D3 to differentiate, in vitro, into forms that are functionally and morphologically similar to macrophages. Macrophages from Vitamin Di-deficient (D^) mice have impaired intlammatory and phagocytic responses (15). The impaired phagocytic response of peritoneal macrophages can be corrected by incubation with 1,25(OH)2D3 (15). Since DBP is the Vitamin D3 transport protein, interaction of DBP with macrophages may be a prerequisite for macrophage differentiation and macrophage activation. Thus, we suggested that serum DBP in a2-globulin serum fraction is a possible serum factor required for macrophage activation. A

141

recent preliminary study (unpubl. data) revealed that anti-human DBP treated human serum and a2-globulin fraction are unable to support activation of macrophages and that cultivation of DDG-treated peritoneal cells in EA medium containing a small amount (01-2-6 ng/mL) of purified human DBP produced a greatly enhanced ingestion activity of macrophages. DBP is known to bind to lymphocyte membranes (16.17), A low level of macrophage activation by DDG treatment and cultivation of peritoneal cell in a serum-free EA medium can be explained by the possibility that a smalt amount of already lymphocyte bound DBP is available for the generation of macrophage activating factor. The conditioned media of individually DDGtreated B and T cells are unable to activate macrophages after 3 h cultivation of adherent cells (3). This suggests that both cell types are involved in transferring activation signal to macrophages. However, cultivation of adherent cells with a mixture of treated B cell conditioned medium and treated T cell conditioned medium produced no significant activation of macrophages. Therefore, stepwise signal transmission among non-adherent cells must have occurred (3). In fact, macrophage activating signal is derived from DDG-trcated B cells and modified by untreated T cells to yield ultimate macrophage activating factor (3). A preliminary study showed that each non-adherent cell type could begin to process signal(s) in a very short period (less than 15 min). This rapid developmental process, therefore, suggests that a serum factor of a2-globulin fraction is modified by pre-existing functions or enzymes of B and T cells in stepwise fashion to yield the macrophage activating factor. This hypothesis is supported by the capability of DDG-treated non-adherent (B and T) cell ghosts to generate macrophage activating factor. Acknowledgements We are much indebted to Dr Sidney Weinhouse for fruitful advice and encouragement throughout this sludy. This investigation was supported in part by USPHS Granl CA06927 and an appropriation from Commonwealth of Pennsylvania lo I.M. and National Science Foundation Oranl DMB-85M651 to N.Y.

REFERENCES Yamamoto. N. and Ngwenya. B. Z. 1987. Activation of macrophages by lysophospholipids and ether derivatives of neutral lipids and phospholipids. Cane. Res. 47: 2008-2013.

2. Yamamoto. N., Ngwenya. B. Z,. Sery, T. W, et al. 1987. Activation of macrophagcs by ether analogs of lysophospholipids. Cane. Immunol. Immunother. 25: 185-192.

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3. Yamamoto. N.. St Claire. D. A.. Homma, S. ctal. 1988. Activation of mouse macrophages by alkylglycerols. inflammation products of caneerous tissues. Cane. Riw. 48: 6044-6049. 4. Snyder.F. and Wood, R. 1969. Alkyl-1-enyl ethers of glycerol in lipids from normal and neoplastic human tissues. Cane. Res. 29: 251-257. 5. Howard. B. W.. Morris. H. P. and Bailey, J. M. 1972. Etherlipids. a-glycero! phosphate dehydrogenase and growth rate in tumors and cultured cells. Cane. Res. 32: 1533-1538. 6. Ngwenya, B. Z. and Yamamoto. N. 1985. Aetivatlon of peritoneal macrophages by lysophosphatidylcholine. Biochem. Biophys. .4cw839: 9-15. 7. Ngwenya, B. Z. and Yamamoto. N. 1986. Effects of inflammation products on immune systems: Lysophosphatidylcholine stimulates macrophages. Cunc. Immunol. Immuiwlher. 21: 10741082. 8. Baumann.W.J, and Mangold, H.K. 1964. Reaction of aliphatic methansulfonates. 1. Synthesis of long-chain glycerol-(l) ethers. J. On^. Chem. 29: 3022-3057, 9. Tyan, M. L. and Ness, D. B. 1971. Mouse blood lymphocytes: in vilro primary and secondary response to two synthetic polypeptides. / Immunol. 106: 289-291. 10. Bianco, C, Griffin, F. M. and Silverstein, S. C. 1975. Studies of the macrophage complement receptors. Alternative of receptor function upon macrophage activation, y. Exp. Med 141: 12781290.

11. Abe. E.. Shiina, Y., Miyaura. C. et al. 1984. Activation and fusion induced by 1,25-dihydroxyvitamin D.i and their relation in alveolar macrophages. Proc. Natl Acad Sci. USA, 81: 71127116. 12. Abe, E., Miyaura, C . Sakagami, H. er al. 1981, Differentiation of mouse myeloid leukemia eells induced by 1,25-dihydroxyvitamin D.i. Froc. Natl Acad. Sci. USA 78: 4990-4994, 13. Muro, S., Gemmel. M. A. Callaham, M. F. ct al. 1983. Control of macrophage cell differentiation in human promyelocytic HL 60 leukemia cells by t ,25-dihydroxyvitamin DT, and phorbal-l 2-myristate. Cane. Res. 43: 4989-4996. 14. Oisson, 1., Goldberg, U. and Ivhed. 1. 1983. Induction ofthe human histocytic lymphoma cell line U-939 by 1,25-dihydroxycholecalciferol. Cane. Res. 43: 5862-5867. 15. Bar-Shavit, Z..NoffD., Edelstein.S. 1981. 1,25dihydroxyvitamin D:i and the regulation of macrophage fusion. Calif Ti.s.\tie Ini. 33: 673676, 16. Constans. J,. Oksman. S, and Vian. M, 1981. Binding of the apo and holo form of the serum vitamin D binding protein to human lymphocyte cytoplasm and membrane by indirect immunofluorescence. Immunol. Lett. 3: 159-162. 17. Machii.T..Kimura, H.. Ueda. E. CM/, 1986, Distribution of Gc protein (vitamin D binding protein) on the surfaces of normal human lymphocytes and leukemic lymphocytes, Ada Hcmatol. 75: 26,