Phospholipid composition of human monocytes and alterations occurring due to culture and stimulation by C3b

Biochimica et Biophysica Acta, 804 (1984) 301-307

301

Elsevier BBA 11315

P H O S P H O L I P I D C O M P O S I T I O N O F H U M A N M O N O C Y T E S AND A L T E R A T I O N S O C C U R R I N G D U E T O C U L T U R E A N D S T I M U L A T I O N BY C3b FRED F. KENNETT a, HARVEY A. SCHENKEIN a,., THOMAS M. ELLIS b, and R. BRUCE RUTHERFORD a,** a Departments of Periodontics and b Microbiology and Immunology, Medical College of Virginia, School of Dentistry, Richmona~ VA 23298 (U.S.A.)

(Received December 20th, 1983) (Revised manuscript received March 20th, 1984)

Key words: Phospholipid composition; Complement component; C3b; (Human monocyte)

The phospholipid composition of human peripheral blood monocytes has not been previously reported, due to difficulty in isolating these cells in a purified state. In this study, monocytes were purified by counterfiow centrifugation without selective adherence, and were characterized with the use of fluorescent monoclonal antibodies to T and B lymphocytes and monocytes by flow cytometry. These platelet-free cell preparations contained less than 5% T cells and less than 3% B cells. Isolated monocytes, which were rapidly frozen after isolation, contained phospholipids (in order of decreasing concentrations) as follows: phosphatidylcholine > phosphatidylethanolamine > sphingomyelin > phosphatidylserine > phosphatidylinositoi > cardiolipin. A small amount of lyso-PC, but no lyso-PE, phosphatidic acid or lyso-PI, was found. The effect of culturing these cells in the presence or absence of a known stimulant of monocyte prostaglandin E and thromboxane release, the C3b fragment of the third component of human complement (C3), was studied with regard to phospholipid composition. Monocytes cultured without stimulant for 24 h contained 3 - 4 % more sphingomyelin than did uncultured cells, and lyso-PC concentrations were consistently elevated. The addition of the stimulant C3b to cultured cells resulted in enhancement of release of immunoreactive prostaglandin E into culture supernatants, without affecting the release of lysosomal enzymes. Analysis of the phospholipid content of cells cultured in the presence of C3b revealed that there was a significant decrease in total PI compared to cells cultured in the absence of C3b, in addition to an increased concentration of sphingomyelin and lyso-PC when compared to freshly isolated cells. These changes occurred in the absence of elevated concentrations of phosphatidic acid. Introduction Cells of the m o n o c y t e s / m a c r o p h a g e lineage are capable of the enzymatic conversion of polyun* To whom correspondence should be sent. ** Current address: University of Connecticut School of Dental Medicine, Department of Oral Biology,Farmington, CT 06032 (U.S.A.) Abbreviations: PC, phosphatidyleholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; C3, third component of human complement; C3b, 180 kDa fragment of C3. 0167-4889/84/$03.00 © 1984 Elsevier Science Publishers B.V.

saturated fatty acids, particularly arachidonic acid to the biologically active eicosanoids thromboxane A 2 and prostaglandin E 2 [1]. Amongst the mononuclear leukocytes, the monocyte (macrophage), with a high concentration of arachidonic acid in phospholipid (over 20%) [2,3], is the principle source of eicosanoids. Most, if not all, lymphocytes do not synthesize prostaglandin or thromboxanes, despite the presence of adequate concentrations of arachidonic acid in their membrane phospholipids [4]. A variety of substances, such as zymosan [5], concanavalin A [6], Ca 2+ ionophore

302 [7], aggregated immunoglobulin [8], immune complexes [9], phorbolesters [10], and fragments of the third component of human complement (C3) [11] have been shown to stimulate macrophage prostaglandin release. It is well established that prostaglandins and thromboxanes are not stored in mammalian tissues, and that the concentration of available unesterified fatty acids in these tissues is insufficient to account for the cell's potential to produce metabolites of arachidonic acid. Therefore, it is generally accepted that the rate-limiting step for the synthesis of these eicosanoids is the release of arachidonic acid from cell phospholipid. It has further been shown that different stimuli induce different pathways of macrophage phospholipid metabolism [7,10]. Until recently, studies of arachidonic acid metabolism and phospholipid composition of human blood monocytes have been hindered by the difficulties in preparing platelet-free cell populations and by the potential alterations that may occur when cells are isolated by selective adherence culturing techniques. We have recently demonstrated that the purified, fluid-phase complement components C3b and iC3b stimulated the release of immunoreactive prostaglandin and thromboxane from human peripheral monocytes that were isolated by counterflow centrifugation [11,12]. This isolation technique provides plateletfree cell preparations that have not undergone an adherence and disadherence phase of purification. The purpose of the present study was to examine the phospholipid composition of well-characterized preparations of purified human monocytes and the changes that occur during culture of these cells for 24 h. In addition, we examined the effects of C3b, a known stimulant of monocyte prostaglandin release, on phospholipid composition and lysosomal enzyme release during culture. Methods

Cell isolation and culture The techniques for the preparation of ceils for culture, isolation of C3b and incubation conditions were as reported previously [6]. Briefly, mononuclear cells collected following centrifugation of citrated blood on Ficoll-Hypaque gradients

were separated into monocyte and lymphocyte populations by counterflow centrifugation. The monocyte population was cultured in medium MCDB 104 [13] in the absence of serum or protein additives. The C3b utilized for this study was prepared by limited trypsin digestion [14] of purified human C3 [15]. The characteristics of such preparations have been previously published [11]. Monocytes isolated by counterflow centrifugation were characterized as described below, and their concentration was determined by microscopic counts and by analysis on a Coulter cell counter. The monocytes were divided equally into three groups, and were diluted to a final concentration of 1 • 1 0 6 cells/ml with culture medium. One group served as a control and was immediately frozen at - 60 o C. The other two groups of (12-24). 106 cells were cultured for 24 h in the presence or absence of C3b (25/~g/ml).

Extraction of phospholipid and analysis After the 24 h incubation, the culture media were removed with a Pasteur pipette, and adherent cells were mechanically harvested. Cells were suspended in 0.1 M KC1 with a glass-Teflon tissue homogenizer and samples were removed for enzyme analyses (see below) prior to extraction for lipids. The noncultured controls were thawed, and the media from these samples and the experimental cultures were clarified by centrifugation in a Beckman 50.1 rotor. All clarified supernatants were saved for analysis of their lysosomal enzyme activity. Corresponding ceils and membrane fragments were extracted for total lipids by the method of Folch et al. [16] as modified by Beckman et al. [17] with re-extraction of the upper phase. 10 vol. chloroform/methanol (2:1) were added per volume of cells. Combined chloroform phases were taken to complete dryness and redissolved in 100 /~1 of chloroform/methanol (9:1). Phospholipids were separated according to the method of Turner and Rouser [18] in the presence of butylated hydroxytoluene. Total phosphorous and individual phospholipid phosphorous were determined according to the method of Bartlett [19]. Enzyme and prostaglandin determinations fl-Glucuronidase and N-acetyl-fl-glucosaminidase were assayed according to the method of

303

Ruth et al. [20]. Lysozyme was assayed using micrococcus lysodeiktius as described by Shugar [21]. I m m u n o r e a c t i v e prostaglandin was determined by a competitive radioimmunoassay as described previously [11]. Samples or PGE 2 standards were incubated with rabbit anti-prostaglandin E-bovine serum albumin and 3H-labeled prostaglandin E 2. After 90 min, goat anti-rabbit IgG plus pre-immune rabbit serum was added to the incubation mixture. Bound radioactivity in the precipitate was determined by liquid scintillation spectroscopy. The commercial rabbit anti-prostaglandin E serum (Miles Laboratory, Elkhart, IN) displayed the following specificity: prostaglandin E2, 100%; prostaglandin El, 70%; prostaglandin A 1, 1%; prostaglandin F2, 5%; prostaglandin B1, 0.6%; and prostaglandin B2, 0.1%.

TABLE I

Characterization of cell populations

clonal antibodies directed against surface antigens of various mononuclear cell types were assessed. The data in Table I show the relative concentrations of T lymphocytes (OKT3- and OKTll-positive cells), B lymphocytes (Bl-positive cells) and monocytes (Macl-, OKM1- and Mol-positive cells). T lymphocyte markers were present on less than 5% of cells in the monocyte preparations, compared to 60% OKT3 ( + ) cells and 76% O K T l l ( + ) cells in unseparated mononuclear cell preparations. Bl-positive cells comprised 2.5% of the

Cell ppopulations were characterized by their reactivity with the following monoclonal antibodies: a-OKT3 and a-OKTll were the kind gift from Dr. T. Tachovsky. Anti-OKM1 was from Ortho Diagnostic Systems, Inc. (Raritan, N J), a-B1 was from Coulter Electronics, Hialeah, FL, and a-Mol and a-Macl were generously provided by F. Todd and T. Mohanakumar, respectively. Cytometric analysis was performed using an Ortho Spectrum II flow cytometer (Ortho Diagnostic Systems, Westwood, MA) equipped with a Iw argon-ion laser, emitting at a wavelength of 488 nm at an operating power of 30 mW. Forward and fight angle (90 o) light scatter was measured using photomultiplier gain settings of 27 and 210, respectively, and an integrator gain setting of 5 for both parameters. Green fluorescence was measured using photomultiplier and integrator gain settings of 540 and 15, respectively. Fluorescencepositive ceils were expressed as the percentage of total cells which showed staining intensities beyond a window containing 99.5% of control stained cells. Open trigger regions were utilized in all analyses, in order to include all cells present in each sample. Results

In order to determine the purity of the monocyte preparations, their reactivities with mono-

FLOW CYTOMETRIC ANALYSIS POPULATIONS PREPARED BY CENTRIFUGATION

OF MONOCYTE COUNTERFLOW

Mononuclear cells were prepared by centrifugation on FicollHypaque gradients. Monocytes were prepared from mononuclear cells by counterflow centrifugation, n.d., not determined. Mononuclear cells Monocytes N u m b e r of (% positive ± S.D.) (% positive ± S.D.) determinations OKT3 OKTll B1 OKM 1 MO 1 MAC1

60.0+ 8.3 75.7+11.9 7.7± 2.8 n.d. n.d. 9.5± 0.0

4.8+ 1.1 2.8-t- 0.3 2.5± 1.5 85.0±10.0 77.9± 0.9 84.2± 0.3

4 4 2 2 2 2

T A B L E II QUANTITATION OF N-ACETYL-fl-GLUCOSAMINIDASE A N D I M M U N O R E A C T I V E P R O S T A G L A N D I N IN SUPERNATANTS FROM MONOCYTES CULTURED FOR 24 H W I T H O R W I T H O U T C3b Total N-acetyl-fl-glucosaminidase activity in freshly isolated monocytes was 0.885+0.166 /~M/106 cells/rain. Data are expressed as the mean + S.D. Control (n = 6)

C3b, 2 5 / ~ g / m l (n = 6)

N-acetyl-fl-glucosaminidase Total activity 0.786 + 0.296 0.708 + 0.159 ( ~ M / 1 0 6 ceUs/min) N-acetyl-fl-glucosaminidase % in supernatants 15.8 +7.7 15.9 +3.7 Prostaglandin E (rig/106 cell) 2.72 + 2.1 9.44 4- 6.2 ~ a Significantly different from control, P < 0.05 as determined by Student's paired t-test.

304 monocyte preparations, compared to 7.7% in the mononuclear cell preparations. The monocyte markers (OKM1, Mol, and M a c l ) were present on 78-85% of cells in the monocyte preparations. Mac!-positive cells comprised 9.5% of the mononuclear cells prior to counterflow centrifugation. These data confirm our previous observations on the purity of these cells, which were shown to be over 90% phagocytic and esterase positive [6]. The remaining mononuclear cells in the monocyte preparations are thought to be null cells or promonocytes. Microscopic analysis and size distribution of monocyte preparation revealed a complete absence of platelets, either free or adherent to the monocyte surfaces. The data in Table II depict the activity of the lysosomal enzyme N-acetyl-fl-glucosaminidase found in clarified supernatants and in detergentsolubilized cell pellets in six experiments. Despite a decrease in the mean total enzyme activity of the cultured cells as compared to that in noncultured cells that were frozen immediately after isolation, no significant differences were noted between cultures exposed to C3b and those exposed to medium alone. Thus, C3b failed to stimulate release of N-acetyl-fl-glucosaminidase under these conditions. Determinations of the enzymatic activities of lysozyme and fl-glucuronidase (data not shown) also failed to demonstrate an effect of C3b on

these lysosomal enzymes. Lysozyme was slightly more membrane associated, with release of approx. 10% of the activity into the supernatant. fl-Glucuronidase was slightly more labile; approx. 20% of the total activity was released into the culture medium. Table II also demonstrates the release of immunoreactive prostaglandin in these six experiments. As was previously noted, C3b-stimulated prostaglandin production 3- to 7-fold when compared to the corresponding controls. Cultured cells produced prostaglandin at low levels (range 0.54-6.0 ng/106 cells), but prostaglandin levels in noncultured cells barely reached reliable limits of detection. In similar preparations, we have shown 5- to 10-times as much thromboxane B2 release as prostaglandin E 2 [12]. Table I I I shows the results of the analysis of the monocyte membrane phospholipids expressed as a percentage of total recovered phosphate. Mean recovery was found to be 93.4+7.1% (range 84-111). For all samples, total phosphates were 6.0-7.5 nmol phospholipid P/106 cells. No significant differences were noted between experimental groups. The various phospholipids are listed according to their relative concentrations in these membranes. PC was always found to be the major phospholipid, followed by PE and sphingomyelin in each of the preparations. These phospholipids accounted for 70-80% of the total phospholipids.

TABLE Ill PHOSPHOLIPID COMPOSITION OF MONOCYTES Data are expressed as percentage of total recovered phospholipid phosphorus+S.D. Remaining phospholipid phosphorus is accounted for by unidentified phospholipid plus material remaining at origin of chromatographs. Data represent results of eight different experiments. Numbers in parentheses represent the number of trials in eight experiments in which lyso-PC was detected. In the two trials in which lyso-PC was detected, it accounted for 0.37 and 0.64~ of the total phospholipid phosphorus. Phospholipid Phosphatidylcholine Phosphatidylethanolamine Sphingomyelin Phosphatidylserine

Phosphatidylinositol Cardiolipin Lysophosphatidylcholine

Uncultured

24-h cultures

cells

control

39.8 + 4.2 30.4 + 3.5 10.9+ 1.1 9.5 + 1.2 6.9 + 1.1 1.4 + 0.8 < 1.0 (2)

36.8 + 3.1 29.6 + 3.4 14.1 + 2.1 a 7.6 + 1.6 6.4 + 1.7 1.2 + 0.5 2.4 + 0.9 (5)

C3b, 25 pg/ml 35.5 + 3.2 a

29.5 + 5.2 16.8 + 3.3 ~

a Significantly different from uncultured cells, P < 0.05 (Student's paired t-test). b Significantly different from cells cultured in the absence of C3b, P < 0.001 (Student's paired t-test).

7.5 + 1.8

5.0 + 1.4 a.b 1.5 + 0.8 3.7 + 1.8 (6)

305 A significant decrease in the major phospholipid, PC, was noted only in cultured cells exposed to C3b. Interestingly, the proportion of sphingomyelin was significantly increased in both cultured cell groups. Analysis of the minor phospholipid species failed to demonstrate any lyso-PE or lyso-PI. As has been noted by others [22], the cardiolipin content of these cells is low, less than 2%, and remains low after 24 h of culture. Lyso-PC was found in trace quantities in two of eight uncultured control samples, but was more consistently found in both cultured cell groups (five of eight and six of eight, respectively). Significant decreases in the content of PI were noted only for C3btreated cells. The decrease was dramatic (over 50%) in four of eight preparations; however, only two preparations yielded any detectable phosphatidic acid after extraction and chromatographic separation. Discussion

This report confirms the utility of counterflow centrifugation as a means to obtain large numbers of highly pure human peripheral monocytes [6]. This procedure allowed us to examine platelet-free monocyte populations, while avoiding the potential problems of using selective adherence during isolation of our starting material [2]. The purity and uniqueness of these cells have been confirmed by enzyme analysis, microscopy, cell-sizing analysis, and now by immunological techniques (Table I). The release of prostaglandins and thromboxanes from leukocytes has been demonstrated to occur in response to a variety of stimuli. Elucidation of the phospholipid source of the precursor fatty acid for eicosanoid production has been an active area of study, and is of particular interest with regard to macrophage immunomodulatory functions that may be mediated by metabolites of arachidonic acid. Although data are available describing the phospholipid composition of certain leukocyte populations and changes that occur during prostaglandin production, to our knowledge our data are the first published which describe the phospholipid content of well-characterized human monocytes. Lipid analysis of human mononuclear

cells reported to this point have been performed on cells containing significant numbers of platelets, lymphocytes and monocytes [3,23] or have failed to provide an individual accounting of all phospholipid species [24]. Thus, these data reflect the relative contribution of contaminating cells to total phospholipid analysis, or provide an incomplete or inaccurate picture of phospholipid composition, when only major phospholipids are described. Our choice to examine changes in total cellular phospholipids rather than pulse-labeled phospholipid pools was made to avoid labeling of selected phospholipids during early maturation of the monocyte. In a recent study of platelet-depleted monocyte cultures, the uptake of radiolabeled arachidonic acid into membrane phospholipid pools has been reported [2]. The data reported did not reflect the composition of the monocyte membrane phospholipids, as they are reported in this study. Freshly isolated monocytes contained phospholipids as follows: phosphatidylcholine > phosphatidylethanolamine > sphingomyelin > phosphatidylserine > phosphatidylinositol > cardiolipin. Lyso-PE and phosphatidic acid were not detectable in the cells, and lyso-PC was occasionally present in minute quantities. However, since changes in phospholipids were measured after 24 h in culture, these data do not rule out the possibility that phosphatidic acid was formed early and metabolized further to species not identified by these methods. The same is true for lyso-PC, since these cells are capable of active reacylation when fatty acyl coenzyme A is available as substrate. Analysis of phospholipid changes due to culture was also performed. It is well known that as monocytes mature in culture, metabolic and structural ~hanges occur [25]. Proliferation of plasma membrane, lysosomes and mitochondria has been shown to occur as early as 24 h after adherence. This has been confirmed microscopically by a number of groups. Biochemical analyses of changes of total and specific activities of organellar marker enzymes also supports these observations [26]. Our study extends these observations by demonstrating that other membrane-associated changes are occurring. The elevation of lyso-PC and sphingomyelin noted in both cultured cell groups may suggest increased membrane

306 turnover in preparation for spreading or maturation. Sphingomyelin is found at high concentration levels in plasma membranes, and to a lesser extent in other organdies [27]. Lyso-PC is one by-product of the action of phospholipase A 2, an initiating step in prostaglandin E production and a means by which the membrane phospholipid can change the fatty acid composition and thus the physical characteristics of the membrane. Lyso-PC is also utilized for de novo synthesis of PC, the major phospholipid of monocyte membranes. The source of arachidonic acid for production of prostaglandin E and thromboxanes in several other leukocyte systems has been found to be PI and PC, through the use of pulse-radiolabeling techniques. Data that exist demonstrating a predominant pathway for generation of arachidonic acid show a variability that appears to depend on the stimulus utilized and the cell challenged, and it is possible that multiple pathways of arachidonic acid release can be involved [5,7,10]. The degradation of PI and PC and the release of arachidonic acid need not be mutually exclusive; a recent report emphasizes that complex interrelationships may exist between these two processes [31]. In addition, release of lysosomal enzymes in some systems has been reported by others to occur concomitantly with PI turnover [7,28]. We have recently reported that C3b stimulates prostaglandin and thromboxane release from monocytes purified by counterflow centrifugation when they are cultured as described in this study [11,12]. Furthermore, C3b is reported to stimulate release of lysosomal enzymes from rodent macrophages [29,30]. The data reported in the present study indicate that C3b stimulates PI turnover without causing increased release of lysosomal enzymes. In agreement with this concept, others have reported that lysosomal enzyme release and prostaglandin production are not necessarily interdependent processes [5,24]. Our data in Table II demonstrate that human monocytes are capable of prostaglandin production. When stimulated by C3b, a 3- to 6-fold increase in the release of immunoreactive prostaglandin was noted. However, no increase in the release of lysosomal enzymes from these cells was noted after stimulation by C3b. The measurements of lysosomal enzymes did not differ significantly in total activity or in the proportion of activity that remained bound or

solubilized for all three enzymes tested. This suggests that, under the conditions reported here, prostaglandin E release and lysosomal enzyme secretion are independent events. We have not studied the influence of cytochalasin B in this system. Examination of the changes in PI for each individual cell preparation from our study alse failed to show any significant correlation between PI turnover and lysosomal enzyme release. When cultured monocytes were exposed to C3b, changes similar to those seen in nonstimulated cultures were noted in their content of lyso-PC and sphingomyelin. In four of the eight C3b-treated cultures the proportion of lyso-PC recovered was slightly greater than that in corresponding cultured controls, while the increase in sphingomyelin was similar for both groups. Also, in four of the eight C3b treated cultures a large decrease in the content of PI was noted. A similar trend in PI depletion was noted by Serhan et al. [32] following stimulation of human neutrophils. It was thus difficult to segregate changes due to normal membrane turnover of phospholipids and phospholipid changes that may be induced by C3b during prostaglandin E and thromboxane production. This includes the complex metabolic changes that the various intermediates of the major phospholipids may undergo. Our data clearly show that relative changes in phospholipid composition may occur due to culturing conditions, and total quantification of major and minor phospholipid must be performed in order to assess accurately the significance of these changes. Our data are consistent with the interpretation that PI is one possible source of arachidonate for prostaglandin and thromboxane production when the monocyte is activated by C3b. However, these data cannot be interpreted to indicate the presence of a receptor triggered phosphatidylinositol cycle in human monocytes. The decrease in PI could be due to enhanced phospholipase A1, C or D activity. Data demonstrating the release of arachidonic acid from phosphatidic acid and its conversion to prostaglandin in response to C3b would be required to suggest an active phosphatidylinositol cycle. Experiments to identify the precise source of monocyte arachidonic acid which is converted to thromboxanes, prostaglandins or other metabolites are in progress.

307

Acknowledgements T h i s w o r k was s u p p o r t e d in p a r t b y G r a n t s D E - 0 5 5 1 2 a n d D E - 0 5 6 2 6 f r o m the N a t i o n a l ins t i t u t e of D e n t a l Research, N a t i o n a l I n s t i t u t e s of Health, and by Biomedical Research Support G r a n t S 0 7 R R - 0 5 7 2 4 . F r e d F. K e n n e t t is r e c i p i e n t of Postdoctoral Training Fellowship T32HL-07244, a n d H a r v e y A. S c h e n k e i n is a r e c i p i e n t of a Research Career D e v e l o p m e n t Award DE-00108, b o t h f r o m the N a t i o n a l I n s t i t u t e s of H e a l t h . T h e a u t h o r s wish to t h a n k Ms. D e b r a Bruce, Ms. Lissa J a c k s o n , a n d Mr. R e n n i e Berry for their e x c e l l e n t t e c h n i c a l assistance.

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