release of galactosyltransferase from peritoneal macrophages during acute inflammation

JOURNAL OF CELLULAR PHYSIOLOGY 124:137-145 (1985)

Release of Galactosyltransferase From Peritoneal Macrophages During Acute

Inflammation K. HOPPER,* W. HOLLISTER, R. DAVEY, AND A. SEMLER

Kolling lnstitute of Medical Research (K. H., W. H., AS. ) and Department of Oncology (R. D.), Royal North Shore Hospital of Sydney, St Leonards, NSW 2065, Australia

Peritoneal cells harvested from mice injected with Salmonella enteritidis or thioglycollate released large amounts of galactosyltransferase (GT), but not sialyltransferase, into their culture supernatants. Maximum release of GT (using ovalbumin as acceptor) occurred from cells harvested 2-4 days after primary injection, but little GT was released from cells elicited by a secondary injection of salmonella or ovalbumin in sensitised mice or during intraperitoneal allogeneic reactions. Enzyme release in culture did not parallel GT levels in serum. Most enzyme was released by large, poorly adherent, macrophage- enriched, Fc receptor-bearing peritoneal cells of low density. Normal monocytes, bone marrow cells, and platelets also produced large amounts, and normal spleen cells or polymorphonuclear leukocytes moderate amounts, of GT. Lymphocytes, dead cells, mast cells, red blood cells, or whole populations of lymph node and thymus cells released very low levels of enzyme. Very little GT was bound to the cell surface and was not passively absorbed from serum or platelets. Release of GT was prevented at 4OC but was not markedly affected by a variety of metabolic inhibitors except pretreatment of the cells with thrombin, which increased release and trypsin which decreased release.

The majority of extracellular complex carbohydrates in animals are synthesised in the Golgi or smooth endo- plasmic reticulum by membrane-bound glycosyltransferases and then exported or expressed on cell surfaces. Ectoglycosyltransferases, particularly galactosyltransferase (GT), have also been detected on external cell surfaces (see review by Shur and Roth, 1975) and in body fluids such as serum, milk and amniotic fluid (Weiser and Wilson, 1981; Kim et al., 1972). The serum enzymes have general properties similar to those of the membrane-bound enzymes except that the soluble activities are not enhanced by detergent (Kim et al., 1972). These enzymes may be involved in cellular communica- tion or adhesion (Shur and Roth, 1975; Dennis et al., 1982; Rauvala et al., 1983); as opsonins and recognition factors of foreign or effete material (Parish, 1977; Roth- enberg, 1978); or in modification, regulation or repair of tissue oligosaccharides.

Serum glycosyltransferases are elevated in humans and animals during acute inflammation (Canonico et al., 1980; Kiang et al., 1978) and in the serum of patients with cancer (Weiser and Wilson, 1981). Serum galactosyltransferase isoenzymes and sialyltransferases have been suggested as markers for the presence of tumours (Weiser and Wilson, 1981; Davey et al., 19841, although the source of these enzymes has not been determined. Hepatocytes (Turco and Heath, 1976) and macrophages (Canonico et al., 1978; Patt and Grimes, 1974) have been suggested as sources of extracellular GT. In this study we have assessed the local production of extracellular GT by different cell populations during acute intraperi-

0 1985 ALAN R. LISS. INC

toneal inflammation induced by different agents. This has been done to establish a basis for interpretation of changes in GT levels in cancer patients and to study the possible functions of these enzymes in inflammatory and immune reactions.

MATERIALS AND METHODS Animals and preparation of peritoneal cells

Details of the intraperitoneal injections and cell-har- vesting procedures are given elsewhere (Cahill and Hop- per, 1982,1984; Hopper and Cahill, 1983). In brief, 8-12- week-old male CBA/H mice were normally injected 3 days before harvest with 2 ml brewer’s thioglycollate broth (TG, BBL, Becton Dickinson, Cockeysville, MD), Corynebacteriurn paru urn (heat-killed [Wellcome Austra- lasia Ltd., Cabramatta], 1.4 mg in 0.2 ml saline) lipopo- lysaccharide (LPS, type B from Salmonella enteritidis [Difco Laboratories, Detroit, MI], 50 pg in 0.2 ml saline) or were infected with Salmonella enteritidis 11 RX (SE, lo6 viable organisms in 0.2 ml saline) either as a primary infection in normal mice or as a secondary infection in mice sensitized intraperitoneally 3 weeks before with 5 x lo5 bacteria. Allogeneic reactions in the peritoneal cavity were produced by injecting lo7 norma lymph node cells from C57BU6 mice into normal CBA H mice. Control mice received normal saline or norma CBA lymph node cells.

Received May 14, 1984; accepted February 4, 1985. *To whom reprint requests/correspondence should be addressed.

138 HOPPER, HOLLISTER, DAVEY, AND SEMLER

Preparation of other cells Bone marrow cells were obtained from the tibias and

femurs of normal mice by inserting a 26-gauge needle into one end of the bone and washing through with 2 ml of warm Hank's balanced salt solution (HBSS). Blood was collected by cardiac puncture, and citrated mononuclear cells were separated from citrated blood on Lym- pho-Paque (Nyegaard and Co., Oslo) and then on a discontinuous gradient of Percoll (see below). Peritoneal neutrophils were induced with 2 ml of TG after 16-20 hours and fractionated to 98% purity by discontinuous density gradients of metrizimide (Vadas et al., 1979). Mast cells were similarly purified to > 95% purity from normal peritoneal washouts. Platelet-rich plasma was prepared from citrated blood by centrifugation at 200g for 5 minutes. This was centrifuged at 3000g for 15 minutes to prepare platelet-poor plasma or platelets as described by Musson and Henson (1979).

Separation of cells 1. Density. Discontinuous gradients of Percoll (Phar-

macia Fine Chemicals, Uppsala, Sweden) in phosphate buffered saline were prepared in 2.5 percentage steps from 45% to 57.5% Percoll. The cells were layered onto the gradient and centrifuged for 30 minutes at 2,800 rpm and 20" in the swingout head of a Mistral 6L centrifuge. The interface cells were washed twice with HBSS and made up to 3 x lo6 with culture medium. In some experiments, gradients of metrizamide were used to separate macrophage enriched exudates (Vadas et al., 1979).

2. Sedimentation velocity (SV). Peritoneal and bone marrow cells were fractionated by SV at unit gravity as described elsewhere (Hopper and Nelson, 1979).

3. Adherence. Peritoneal cells (0.3 x lo6) in 0.2 ml Dulbecco's modified Eagle's medium (DME, Gibco, Grand Island, NY) supplemented with 100 pg.ml streptomycin and 100 U/ml penicillin, containing 10% fetal calf serum (FCS, Flow Laboratories, Annandale, Australia) previously heated for 30 minutes at 56"C, or 1% bovine serum albumin (BSA, Cohn Fraction V, Commonwealth Serum Laboratories, Parkville, Australia) were incubated in 96-well flat-bottom plates for 90 minutes a t 37"C, and the adherent cells washed three times with warm medium.

4. Rosetting. Fc receptor-bearing cells were rosetted with sheep red blood cell (SRBC) coated with subagglu- tinating concentrations of rabbit anti-SRBC serum (Par- ish and McKenzie, 1978) and separated from unbound cells by centrifugation into Ficoll-Hypaque (Pharmacia) of density 1.086 gkc. The cells were washed, and the SRBC were lysed by brief water shock treatment and resuspended in DME.

5. Treatment with antisera to Thy 1.2. The cells were incubated with a 1/100 dilution of a monoclonal anti- serum (titre 80,000, Australian Monoclonal Develop- ment, Artarmon) as previously described (Cahill and Hopper, 1984). After incubation with complement, the cells were washed twice with DME.

Cell incubations Cells were cultured in DME, and the supernatant was

diluted and supplemented with MnClz before assay. No major differences in results were observed using other

buffers without phosphate salts, such as HEPES or Tris, for short incubations. Serum supplements were not included, since, although the serum GT can be inactivated by heating for 30 minutes at 56"C, enzyme activity was slowly regenerated on storage (at -20°C to 4"C), mak- ing interpretation of the results difficult. Addition of 1% BSA or 5% and 10% heat-treated fetal calf serum, however, did not alter enzyme release over 3 hours. The cells (3 x lo5 from 3-6 mice) were incubated either in sterile polypropylene conical tubes (Eppendorf Geratbau, Ham- burg, W. Germany) or in flat-bottom, 96-well plates (Fal- con Microtest I1 plates) in 150 p1 medium. Results were the same using the tubes or plates. At the end of the incubation period the tubes or plates were centrifuged and 60 pl of the supernatant gently removed for assay.

In some experiments, cells in the culture media were frozen and thawed three times by immersing the tubes in liquid nitrogen and warming quickly at 37"C, result- ing in death of all cells. Cells were sonicated for 20 seconds on setting 40 of a Biosonik Instrument. The following reagents were included in some cultures: cycloheximide, puromycin, cytochalasin B, colchicine, adenosine diphosphate (ADP), dibutyrl cyclic AMP, dibutyrl cyclic GMP, ethyleneglycol-bis-(P-aminomethyl ether) N,N'-tetraacetic acid (EGTA), ethylene diamine tetraacetic acid (EDTA), indomethacin, prostaglandins El and Ez (Sigma Chemical Co., St. Louis, MO). D- galactonolactone was kindly provided by Dr. C. Marsh, New South Wales Institute of Technology, Gore Hill, Sydney). Following treatment with EDTA or EGTA, the supernatants were dialysed twice with medium. Other reagents were not removed before assay and were found to have no effect on GT activity. Cells were pretreated with trypsin (bovine pancreatic) or thrombin (bovine, 1, 10, or 100 pg per 1.5 x lo6 cells in 500 pl DME, Sigma) for 30 min at 37"C, 50 pl freshly heat-inactivated FCS added, and the cells were washed three times with cold medium.

In preincubation experiments, Eppendorf tubes were set up with combinations of resident or thioglycollate- induced cells and platelet-poor or platelet-rich plasma, serum, or washed platelets, incubated for 2 hours at 37°C and washed three times by low speed centrifugation. The tubes were incubated for a further 3 hours and the GT in the supernatants determined.

Gaiactosyltransferase assay GT activity was measured in 60-pl aliquots of culture

supernatants, cell extracts or diluted serum as described previously (Davey et al., 1983) by adding 90 p1 of reaction mixture in water containing 1.8 mg ovalbumin (Sigma) as exogenous acceptor, 1.5 pCi UDP-[3H]-galactose (The Radiochemical Centre, Amersham; specific activity 5-20 Ci/mmole), 2.4 nmoles UDP-galactose (Sig- ma), and 3 p moles MnC12. The mixture was incubated for 1 hour at 37°C in a gently shaking water bath, and then 20 pl of 0.8 M EDTA was added to stop the reaction. A 60-p1 aliquot of the reaction mixture was spotted onto glass-fibre discs, dried, and washed repeatedly with cold 10% trichloroacetic acid (TCA) and ethanol to remove unreacted UDP-[3H]-galactose. The amount of 3H-galactose transferred to ovalbumin was determined. Enzyme activity in a standard sample of normal human serum was used as a positive control.

GALACTOSYLTRANSFERASE FROM MACROPHAGES 139

Cell-associated endogenous acceptor activity was measured by incubating the cells, either as adherent mono- layers or dispersed in tubes, with the enzyme reaction mixture for 60 minutes. The cells were washed twice with warm medium and solubilised by the addition of 0.1% sodium dodecylsulphate (SDS) and counted. All GT activity is expressed as the cpm incorporated per 100 pl of supernatant obtained from 3 x lo5 cells or per 100 pl of 3 x lo5 solubilised cells unless stated otherwise.

Sialyltransferase (ST) assay The method was similar to that used for GT. The

incubation mixture contained 0.9 mg asialofetuin, 0.5 pCi CMP-[3H]-sialic acid (18.9 Cdmmole, New England Nuclear, Boston, MA) and 30 pM CMP-sialic acid (Sigma) in 0.05 M imidazole buffer, ph 7.0.

Lactate dehydrogenase (LDH) and P-D-glucuronidase activities

LDH in culture supernatants was assayed by the fluorimetric method of Lowry et al. (1957) and P-D-glucuronidase by the method of Mead et al. (1955).

Galactosidase activity a- and 0-galactosidase activities were assayed in cul-

ture supernatants a t pH 4.0 and 7.2 as described elsewhere (Paigen et al., 1978; Lusis et al., 1977). The results are expressed in arbitrary units based on the number of nmoles of 4-methylumbelliferone converted per ml of cell supernatant per hour a t 37°C.

RESULTS Release of GT by resident or thioglycollate induced

peritoneal cells Normal resident peritoneal cells released very little

GT over 1 ,3 , or 20 hours in culture. TG induced peritoneal exudates released large amounts of GT on a per cell basis, which reached a maximum 2 days after injection (Fig. 1) and returned to normal levels after 6-10 days. There was an initial increase and decrease in neutrophils over the first 16 hours (date not shown), and the number and percent of macrophages migrating into the peritoneal cavity after TG injection was maximal at days 2 to 3 and remained high to day 8. The ability of the cells to release enzyme did not correspond with the presence of neutrophils, macrophages, lymphocytes, or mast cells (identified by morphology) but coincided with the initial increase of monocytelmacrophages in the peritoneal cavity.

High levels of GT activity were also detected in the cell-free fluid prepared from the peritoneal washout (3 ml: for thioglycollate-induced exudates, 14,222 f 1127 cpm in 100 p1 fluid compared with 7,207 f 389 cpm released from 3 x lo5 washed cells over 3 hours; for resident cells, 34,256 & 3,159 and 286 f 344 cpm, respectively). The activity in the peritoneal fluid was not reduced by centrifugation at 105,OOOg (see below).

Serum GT activity of the injected mice also increased during the inflammation, but there was no simple relationship between these enzyme levels and the GT released by cultured cells from the same mice (Fig. 1).

Effect of other peritoneal stimulants on GT production

When mice were given a single intraperitoneal injection of SE instead of TG, a similar time course of GT

release was obtained, although in this case the number of macrophages increased more gradually up to 6-8 days. When mice were given a secondary intraperitoneal challenge with SE, 21 days after a primary infection the number of cells, particularly macrophages, in the exudates was also high but with maximum at 2-3 days, and the bacteria were cleared much more rapidly than in the primary infection (Cahill and Hopper, 1982). Perito- neal cells from these mice released low levels of GT in culture.

A primary or secondary injection of ovalbumin into normal or sensitized mice or the initiation of an allogeneic response in the peritoneal cavities of normal mice caused only moderate or no increase in activity released per cell (Table 1). More GT was released by cells obtained 4 days after intraperitoneal injection of C. parvum, but injection of LPS (50 pg per mouse) resulted in reduced amounts of enzyme release compared with cells from untreated or saline injected mice. In all of these exudates, the number of mononuclear cells increased to a maximum at 4-6 days, and macrophages were the predominant cell type. However, the macrophages in TG or SE exudates at 2 and 4 days were more vacuolated than other populations.

Characterisation of the secreting cell from thioglycollate exudates

Macrophages were enriched by adherence in 96-well flat-bottom plates in the presence of 1% BSA or 10% fetal calf serum which had been heated to 50°C for 30 minutes. After vigorous washing, more than 97% of the cells incubated in serum were identified as macrophages by morphology and their ability to ingest latex particles. On a per cell basis these cells released much less enzyme (935 + 115 cpm) than the nonadherent cells (3,866 & 76 cpm) or the whole population (4,775 k 242 cpm), although the characteristics of secretion were the same in that production increased linearly with cell number and incubation time, and most enzyme was secreted and not cell bound and depended upon the presence of exogenous acceptor (data not shown). The adherence step did not appear to interfere with the enzyme release because the nonadherent cells washed from the wells accounted for the remaining GT released by the whole population. If the exudate cells were induced by a single injection of SE rather than TG, the adherent population secreted more GT, but these cells were not as effective as the nonadherent cells.

When cells collected 3 days after TG injection were fractionated on discontinuous density gradients of Per- coll, the macrophage-enriched fractions at the top of the gradient secreted most GT (Table 21, while the other fractions containing lymphocytes, neutrophils, and dead cells secreted very little GT. Similarly, using discontinuous gradients of metrizamide, most activity was released by cells in the 18-20% metrizamide interface containing macrophages (data not shown). In both separations GT secretion in all fractions was greatest for the nonadherent cells.

In sedimentation velocity separations, fractions of SV above 6 mm/h containing medium to large cells and enriched for macrophages ( > 98%) released most enzyme at 37°C (maximum release, 4,600 + 800 cpm in SV 7.5- 10.0), but again the adherent cells from each of these fractions were less active (1,108 & 80 cpm). Significant amounts of GT (3,088 k 272) were also released by cells


0

2 0 i c

I 1 1 I I I I I 1 1 I

0 2 4 6 8 '0 0 2 4 6 8 10

Time a f t e r in ject ion ( d a y s )

Fig. 1. Peritoneal cell numbers and the GT production by peritoneal cells and GT in sera following single injections of TG (.) or S. enteritidis (0) or a secondary infection of S. enteritidis (A). The cells or sera were collected at the times shown. Panel a shows the GT activity released from cells in culture for 3 hours and the broken lines show the activity released from cells held on ice for 3 hours. Panel b shows

the GT activity in 10 ~1 of serum. Data points show the means and SD of triplicate assays performed on sera and cells from three individual mice per group at each time point. Panel c shows the number of macrophages identified by morphology; panel d shows the total number of cells harvested per mouse at each time point.

TABLE 1. Effect of different Deritoneal stimulants on release of GT bv cultured cells

GT activity released from cells harvested on- Stimulant Day 2l Day 4' Day 6l

Saline2 ND3 962 + 762 ND Thioelvcollate ND 3.354 + 927 ND I-

Corynebacterium parvum ND 2;638 7 771 ND LPS ND 139 k 805 ND Normal mice + ovalbumin 1,435 k 79 1,626 + 284 1,269 k 117 Sensitized mice + ovalbumin 1,227 f 81 1,333 k 106 1,114 + 133 C57BY6 lymph node cells 1,077 k 106 1,168 + 284 1,061 + 317 'Cells were cultured in triplicate for 3 hours, and the mean f SD of GT activity was determined. 'GT released by normal resident cells was 786 f 174 cpm. 3ND = not determined.

in fractions of SV below 3 mm/h containing the majority of dead cells and lymphocytes. Enzyme was also released by these cells when held on ice for 3 hours, and it is likely that this activity is due to dead cells or debris which had not sedimented.

Seventy percent of the peritoneal cells were separated as Fc receptor bearing and most of the secretory activity was depleted with removal of this fraction (3,544 70 before rosetting and 1,228 & 108 cpm after separation per 0.3 x lo6 cells). Treatment of the cells with anti-Thy 1.2 serum and complement did not deplete the popula-

tion of secreting cells nor significantly increase enzyme activity, demonstrating that T lymphocytes were not a source of the enzyme.

GT release by other cells The data suggest that the secreting exudate cells are

recently recruited but poorly adherent macrophages. Mononuclear cells separated from peripheral blood of untreated mice using Lympho-Paque and Percoll density separations and comprising 38% monocytes also released high activity over 3 hours (Table 3). Normal,

GALACTOSYLTRANSFERASE FROM MACROPHAGES

TABLE 2. Release of GT from TG cells following separation on discontinuous gradients of Percoll

Fraction UnseDarated 1 2 3 4 5

141

47.5150 50152.5 > 52.5 Percoll concentration - 45 45147.5 GT released'

Whole cells 1,554 ? 117 6,648 k 287 1,463 f 156 814 ? 84 613 f 93 600 k 40 Adherent cells 570 f 92 643 i 27 630 i 42 532 f 32 537 k 40 ND

Percent cell types' Macrophages 85 90 75 59 0 0 Lymphocytes 6 2 15 26 40 10 Granulocytes 9 8 10 15 60 90

Percent dead cells3 10 6 12 18 2 7. All

'The whole or adherent cells were incubated for 3 hours at 3 7 T , and the supernatant fluid was assayed for GT. *Percentage of different cell types identified in May Griinwald Giemsa-stained slides. 'Percent dead cells identified by trypan blue exclusion. Total viable cell recovery was 76% of the starting population

TABLE 3. Release of cralactosvltransferase bv different cell DoDulations

Cell ooDulation

GT activity released from cells after-

3 hours' 20 hours'

Normal resident peritoneal cells 747 k 98 1,052 k 47 Thioglycollate-induced peritoneal cells 4,775 k 242 22,033 k 1,700 Bone marrow cells 12,819 ~f: 375 38,762 f 5,420 Spleen cells 1,906 I 53 5,064 f 461 Lymph node cells 109 I 125 1,670 f 30 Thymus cells Blood mononuclear cells' 4,748 + 648 6,745 + 1,020 Red blood cells 6 k 136 ND4 Polymorphonuclear leukocytes (99%) 2,244 +_ 100 4,173 k 108 Mast cells (90%) 40 21 66 k 21 platelets3 2,160 +_ 191 ND 'Expressed as cpm per 100 pl per 3 x lo5 cells. 'Comprising 38% monocytes, 60% lymphocytes, and 2% other cells. 3Calculated for 3 X lo5 platelets from cell dose-response curves over the range 106-108 cells. 4ND = not determined.

69 k 165 215 f 201

unfractionated bone marrow cells released more and spleen cells released less enzyme on a per cell basis than the peritoneal exudate cells. Maximum activity occurred among the macrophage-enriched spleen cells of lowest density, but adherent spleen cells released much less GT than the nonadherent cells or the whole population. Lymph node and thymus cells secreted less than spleen cells over 3 or 20 hours. To see if the lack of activity among the thymus cells was due to the presence of an inhibitor, thymus cells were cocultured for 3 hours at ratios of 1:l or 1:lO with TG-induced peritoneal exudate cells, and the supernatant enzyme activity was determined. The GT activity released from the TG cells (6,943 200 cpm) or thymus cells (40 +_ 30 cpm) at 0.3 x lo6 was not altered in the mixed culture (7,171 & 213 cpm at 1:1, 7,310 146 cpm at 1:lO).

TG-induced, peritoneal neutrophils enriched by density gradient fractionation released moderate amounts, whereas normal peritoneal mast cells and red blood cells released no detectable enzyme. Purified platelets, incubated in DME, released GT, and contamination by these cells would contribute to the enzyme released in cultures of monocytes and possibly, to a less extent, spleen cells. However, no free platelets were seen in any of the peritoneal populations.

Characteristics of enzyme release by TG-induced cells Enzyme production by TG-induced cells increased lin-

early with cell number up to 0.4 x lo6 per well when incubated for either 1 or 3 hours (Fig. 2A) and increased linearly with time up to 4 hours (Fig. 2b). There was a more gradual and variable increase in release at later

time points (compare Fig. 2b and Table 3). Under these culture conditions the enzyme activity in preformed cell- free supernatants decreased by about 30% over 20 hours. Almost all of the extracellular GT activity was free in the culture supernatant, and little could be attributed to cell surface enzyme when that released during the enzyme assay was subtracted. Low levels of GT were released if the cultures were kept on ice for up to 20 hours or during cell preparation at 0-4°C. Enzyme activity was also negligible at 0-4°C. In the absence of exogenous ovalbumin acceptor, only low amounts of la- belled substrate were incorporated into TCA-precipita- ble material in the supernatant (Fig. 2a). Substrate was also incorporated into the cells during incubation but this was less than 5% of that bound to the ovalbumin and this did not increase markedly if ovalbumin was included in the cell culture.

Effects of cell disruption on release of GT Dead or fragile cells may release GT in culture, and

this would contribute to the observed activity. Most cell- associated GT is believed to be membrane bound (Shur and Roth, 1975). To study this aspect further, cells were disrupted prior to enzyme separation and assay. The total GT activity detected after three-cycle freezing and thawing of the cell incubation mixture was the same from uncultured cells as those incubated for 20 hours (Fig. 3). In this time the level of activity in the cell supernatant (after centrifugation of the lysate at 200g) was identical to that released in culture by intact cells. The activity released from cells that had been washed after culture and then lysed decreased, suggesting trans-


6

5

0

z . 4

E X

0 - 3 - .- > 0 m

.- e

I - 2 0

1

0 (

a r b

0.2 0.4 0 5 10 15 20 Number of cells per well ( ~ 1 0 ' ) Time of incubation (hr)

Fig. 2. a. Effect of cell number on the release of GT by TG-induced cells in culture at 37°C. After 3 hours the GT activity in the cell-Gee supernatant (0) or the washed cell pellet ( W ) was determined in the presence of ovalbumin acceptor. Enzyme activity of the cell supernatant in the absence of acceptor (0) and the uptake of label by the cells

Hours in culture

Fig. 3. Effect of freezing and thawing on release of GT from TG cells, At time intervals the cells were lysed and GT was determined (i) in the total lysate mixture (GA-1, (ii) in the supernatant from the lysed mixture after centrifugation at 2OOg (W), (iii), in the lysate after the cells were washed < X 2) first and then frozen and thawed (-A -1 and (iv) in the culture supernatant of intact cells (GO-). Data points show the means and SD of triplicate cultures.

fer of free GT from the cell to culture fluid. Cell viability decreased from 84% to 83% and 76% during the 3- and 20-hour culture, and all cells were dead and partially disrupted after the freezing and thawing. Freezing and thawing had no effect on GT activity in preformed culture supernatants.

in the presence (A) or absence ( A ) of acceptor is also shown. Data points represent the means of single assays of triplicate cultures. b. Time course of release of GT from 3 x lo5 TG-induced peritoneal cells in culture at 37°C (0). The activity of preformed supernatant enzyme in culture in the absence of cells is also shown (0).

To determine whether the GT released from either TG cells in culture over 3 hours or from freezing and thawing was membrane bound, the cell supernatants were centrifuged at 105,OOOg for 30 minutes. The GT activity in the centrifuged samples was reduced by 20-40% compared with the uncentrifuged controls. Addition of Tri- ton X-100 (0.05,0.1, and 0.5% final concentration) to the centrifuged supernatants from cultured or freeze-thawed cells did not change the activity of the enzyme, indicating that the released, nonpelletable enzyme was proba- bly not membrane associated. If the cells were sonicated instead of frozen, more GT was released, but this was now pelletable at 105,OOOg and detergent sensitive.

More GT was released from TG cells after solubilisa- tion with 0.5% Triton X-100 for 30 minutes a t 37"C, and this increased following cell culture a t 37°C (60,092 k 1,684 cpm at 3 hours to 84,287 f 1,140 cpm at 20 hours compared with 47,019 f 3083 cpm for cells held on ice for 3 hours). The enzyme was not removed by centrifugation of the lysate at 105,OOOg. Normal resident cells released low amounts of GT on incubation (494 f 177) and after freezing and thawing (1,157 f 859) but high levels after disruption with Triton X-100 (17,945 f 1037).

Effect of metabolic inhibitors and proteases on release of GT

Protein synthesis and microtubule or microfilament functions appeared not to be important in enzyme release by TG-induced cells over 1 or 3 hours, since puromycin, cycloheximide, cytochalasin B, and colchicine at concentrations from lop7 to lop4 M in the culture did not inhibit secretion by more than 15% of control values. Other agents also known to affect platelet function (ADP, indomethacin, aspirin, PGEI, PGE2, dibutyrl cyclic


AMP, or dibutyrl cyclic GMP) had no apparent effect on enzyme release when they were included in the culture at M. However, EDTA and EGTA over a range of concentrations increased the release in culture (1.7- and 1.5efold for EDTA; 1.9- and 2.0-fold for EGTA at and M, respectively). Pretreatment of the cells with trypsin for 30 minutes at 37°C eliminated subsequent release (Table 4). Similar treatment of the cells with thrombin, however, caused increased release.

To determine if passive adsorption of GT from plasma or platelets contributed to the releasable enzyme, TG- induced cells were preincubated for 2 hours a t 37°C with undiluted platelet-poor plasma, platelet-rich plasma, or purified platelets. After washing the cells three times to remove free platelets and plasma, there was no net difference among the samples in the amount of GT released during subsequent incubation for 3 hours (Table 5). Similar results were obtained using normal mouse serum or normal resident peritoneal cells in the preincubation.

Galactosidase, LDH, and 0-glucuronidase activities in the culture supernatants

To test whether release of a- or P-galactosidases could contribute to the lack of activity by some populations, the cell supernatants from 3-hour cultures were assayed for a- and P-galactosidase activity at the pH optima for these enzymes and at pH 7.2, which is used in the GT assay. There was no significant a- or P-galactosidase activity a t pH 7.2. At lower pH some samples showed strong activity, but this did not correlate with low levels of GT in the samples. When an inhibitor of P-galactosidase (D-galactonolactone) was included at 20, 5, and 1 mM in the assay mixture for GT, there was also no increase in activity.

In another experiment 1- and 2-hour culture supernatants were assayed for LDH, 0-glucuronidase, and GT activities. Throughout the incubation, there was an increase in the ratio of GT to LDH (2.62 to 4.13) and in the ratio of GT to 0-glucuronidase (4.57 to 6.22). There was no detectable decrease in cell viability in this time.

TABLE 4. Effect of trypsin and thrombin treatment on the ability of TG cells to release GT

Treatment Dose (pg) GT activity' Viability2

None 1,348 f 39 85 Trypsin 100 110 f 65 78

10 402 f 127 87 1 1.200 + 408 92

~~ ~ - Thrombin 100 2,212 k 244 79

10 1,375 f 195 86 1 1,265 k 234 86

'Expressed as cpm per 100 pl of supernatant from 3 X lo5 cells. 2Determined by trypan blue exclusion after pretreatment.

TABLE 5. Effect of preincuhation of the TG cells with plasma or platelets on the release of GT

GT activity, tubes plus

Treatment Tubes alone cells Activity added'

Platelet-poor plasma 1,800 f 306 3,587 f 673 280,612 f 9,537 Platelet-rich plasma 1,150 f 190 2,561 k 493 229,265 f 6,768 Platelets (107/tube) 165 + 30 1,389 f 37 90,303 rt 7,957

No addition 0 f 0 1,444 f 357 o + o

'GT activity contained in the plasma or platelets added to the tube.

Release of sialyltransferase Normal mouse peritoneal cells and whole or adherent

TG-induced exudate cells incubated for 1 or 20 hours released no detectable ST, nor did they have ST on the cell surface. There was also no apparent ST production by TG-induced polymorphonuclear leucocytes in culture.

DISCUSSION Release of GT by cells in culture appeared to be asso-

ciated with an early local response to the inflammatory stimulus. Increases in the serum GT levels did not parallel the amounts of locally secreted enzyme, and the activity of the latter may be restricted to the site of inflammation.

Most enzyme was released from peritoneal cells 2-3 days after a single injection of TG or SE, when there was maximum immigration of macrophages and the secreting cell cofractionated with macrophages but not other cell types in density and sedimentation velocity separations and Fc receptor rosetting. However, the secreting cell was poorly adherent, and there was no increased release from cells following secondary or cellular immune responses, although increased numbers of macrophages were also recruited into the peritoneal cavities. We suggest that a subpopulation of macrophages or another similar but as yet uncharacterised cell type may be induced in acute inflammatory reactions and be re- sponsible for the release of most of the GT. Since blood monocytes released large amounts of GT and enzyme production by peritoneal cells occurred at the same time as the influx of new macrophages into the peritoneal cavity, GT release may be characteristic of immature macrophages. Macrophage chemotaxis and labelling following i.v. thymidine injection was high in the same populations as those releasing GT (Hopper and Geczy, 1980; Hopper, unpublished observations) indicating that they were possibly recently derived from blood monocytes. Release of GT did not parallel tests for other macrophage functions, for example, tumoricidal or bactericidal activities, lymphokine-induced procoagulant activity and secretion of PGE and interleukin 1 (Cahill and Hopper, 1981; 1984; Hopper and Cahill, 1983; Hop- per, unpublished observations). Others have described transitory macrophage populations in these exudates (see, for example, Beelen and Walker, 1983) but a relationship between them and GT release is not known.

Platelets are also a rich source of GT, and this may contribute significantly to the normal serum pool of GT. Platelets can adhere to other cells, particularly neutrophils (Payne, 1981), but also monocytes (Musson and Henson, 1979; Perussia et al., 1982); and adhered or ingested platelets may be an original source of the released enzyme. A similar mechanism has been suggested previously for macrophage secretion of neu- trophil-derived elastase (White et al., 1982). From our experiments, this is unlikely for the following reasons. First, no platelets were seen by phase contrast micros- copy or in stained cell preparations of the peritoneal cells. Second, with the exception of thrombin, agents promoting or inhibiting activation of platelets did not affect release of GT from the cells. Third, TG-induced or normal resident cells did not passively adsorb platelets or plasma GT during preincubation for 2 hours at 37°C to be released on subsequent culture. However, platelets may adhere and be ingested by selected monocytes dur-


ing passage through the blood to the inflammatory site, and such a relationship between macrophages and platelet products remains to be explored.

Dead or dying cells are also a potential source of GT in culture, but based on our experiments, these were not believed to make a major contribution in these cultures. There was a lack of coincidence of the major source of GT with dead cells in fractionation studies, time courses, or by comparison of different organ populations, and with LDH and 0-glucuronidase activities in culture supernatants. The amount of GT released in culture was far greater than that expected from the number of dead cells counted and the amount of enzyme available following cell disruption. Freezing and thawing released about a quarter of the activity available on totally dis- rupting and solubilising the cells with Triton X-100. Total enzyme measured after freezing and thawing of cells cultured for varying times was constant, and its distribution between the cell and supernatant fractions suggested the presence of “free” GT that was released in culture or on freezing and thawing.

From a variety of experiments involving metabolic inhibitors or cell disruption, it appears that the GT is not rapidly synthesised during 3- or 20-hour incubation. Pretreatment of the cells with trypsin abolished subsequent GT release, whereas thrombin, which is known to stimulate some cell functions (Chen et al., 1976; Hopper and Geczy, unpublished observations) enhanced release.

Furthermore, about 30% of the activity released in culture or on freezing and thawing was pelletable by centrifugation at 105,OOOg. In general these results suggest that the enzyme is shed from the cell surface, possibly within or on vesicles. Canonico et al. (1978) have studied the subcellular localisation of GT in macrophages and found that it was not restricted to the Golgi apparatus nor could it be dissociated from the plasma membrane marker, 5‘ nucleotidase. Our work is in agreement with the location of some GT on or near the plasma membrane that is available for export. Further experiments using cell subfractionation and membrane markers are required to contirm this.

The function of the extracellular enzyme is not known, but many effects of modification of extracellular carbohydrates are potentially important. There is increasing evidence that the binding of macrophages to bacteria (Freimer et al., 1978) and other structures (Monsigny et al., 1983) occurs via recognition of carbohydrates, and this may be dependent on the stage of activation of the cell (Imber et al., 1982). The binding of galactose- and N- acetylgalactosamine-terminating oligosaccharides to hepatocytes via specific lectinlike receptors is also well documented (Bridges et al., 1982). Extracellular glycosyltransferases may be opsonins, or they may modify carbohydrate side chains on effete or foreign materials so that they are recognised by these cells and removed from the circulation (Winterburn and Phelps, 1972). The presence of high levels of GT in sera of cancer patients may block or divert receptors on immune cells or alter immunosuppressive agents such as orsomucoid. From a practical aspect in tumour diagnosis, local production of GT by nontumour cells may account for some change in serum GT levels. However, its general use in this area must await more detailed analysis of isoenzyme patterns and substrate or acceptor specificities.

ACKNOWLEDGMENTS We thank Jane Cahill and Roselle Harvie for technical

assistance, Drs Carolyn Geczy and D.S. Nelson for help- ful discussion and Mary Cass for typing the manuscript.

The work was supported in part by The Bill Walsh Cancer Fund, The New South Wales State Cancer Coun- cil and a Program Grant from the National Health and Medical Research Council.

LITERATURE CITED Beelen, H.J., and Walker, W.S. (1983) Dynamics of cytochemically

distinct subpopulations of macrophages in elicited rat peritoneal exudates. Cell. Immunol., 82~246-257.

Bridges, K., Hartford, J., Ashwell, G., Klausner, R.D. (1982) Fate of receptor and ligand during endocytosis of asialoglycoproteins by iso- lated hepatocytes. Proc. Natl. Acad. Sci. USA, 79t350-354.

Cahill, J.M., and Hopper, K.E. (1982) Immunoregulation by macrophages: Differential secretion of prostaglandin E and interleukin 1 during infection with Salmonella enteritidis. Cell. Immunol., 67.229- 240.

Cahill, J.M., and Hopper, K.E. (1984) Immunoregulation by macrophages III. Prostaglandin E suppresses lymphocyte activation but not macrophage effector function during SaZmonella enteritidis infection. Int. J. Immunopharmacol., 6r9-17.

Canonico, P.G., Beaufay, H., and Nyssens-Jadin, M. (1978) Analytical fractionation of mouse peritoneal macrophages: Physical and bio- chemical properties of subcellular organelles from resident (unsti- mulated) and cultivated cells. J . Reticuloendothel. Soc., 24t115-138.

Canonico, P.G., Little, J.S., Powanda, M.C., Bostian, K.A., and Beisel, W.R. (1980) Elevated glycosyltransferase activities in infected or traumatised hosts: Non-specific response to inflammation. Infect. Immun. 29:114-118.

Chen, L.B., Teng, N.N.H., and Buchanan, J.M. (1976) Mitogenicity of thrombin and surface alteration of mouse splenocytes. Exp. Cell. Res., 101:41-46.

Davey, R.A., Bowen, R., and Cahill, E.J. (1983) The analysis of soluble galactosyltransferase isoenzyme patterns using high resolution aga- rose isoelectric focussing. Biochem. Int., 6t643-651.

Davey, R.A., Harvie, R., Cahill, E.J., and Levi, J.A. (1984) Serum Galactosyltransferase isoenzymes as markers for solid tumours in humans. Eur. J. Cancer Clin. Oncol., 2Ot75-79.

Dennis, J., Waller, C., Timpl, R., and Schirrmacher, V. (1982) Surface sialic acid reduces attachment of metastatic tumour cells to collagen type IV and fibronectin. Nature, 3OOt274-276.

Freimer, N.B., Ogmundsdottir, H.M., Blackwell, C.C., Sutherland, LW., Graham, L., and Weir, D.M. (1978) The role of cell wall carbohydrates in binding of micro-organisms to mouse peritoneal exudate macrophages. Acta Pathol. Microbiol. Scand. @), 86:53-57.

Hopper, K.E., and Cahill, J.M. (1983) Immunoregulation by macrophages 11. Separation of mouse peritoneal macrophages having tumoricidal and bactericidal activities and those secreting PGE and Interleukin 1. J. Reticuloendothel. Soc., 33t443-456.

Hopper, K.E., and Geczy, C.L. (1980) Characterisation of guinea pig macrophages I. Mobility and maturation of peritoneal cells following inflammatory stimuli. Cell. Immunol., 56t400-414.

Hopper, K.E., and Nelson, D.S. (1979) Specific triggering of macrophage accumulation at the site of secondary tumor challenge in mice with concomitant immunity. Cell. Immunol., 47t163-169.

Imber, M.J., Pizzo, S.V., Johnson, W.J., and Adams, D.O. (1982) Selec- tive diminution of the binding of mannose by murine macrophages in the late stages of activation. J . Biol. Chem., 257:5129-5135.

Kiang, D.T., Kennedy, B.J., and Edstrom, R.D. (1978) Levels of blood group synthetic enzymes in human colonic carcinoma. Proc. SOC. Exp. Biol. Med., 157t411-414.

Kim, Y.S., Perdomo, J., and Whitehead, J.S. (1972) Glycosyltransfer- ases in human blood I Galactosyltransferase in human serum and erythrocyte membranes. J. Clin. Invest., 51t2024-2032.

Lowry, O.H., Roberts, N.R., and Kapphahn, J.L. (1957) The fluorimetric measurement of pyridine nucleotides. J . Biol. Chem., 224t1047- 1064.

Lusis, A., Breen, G.A.M., and Paigen, K. (1977) Nongenetic heteroge- neity of mouse 6-galactosidase. J. Biol. Chem., 2524613-4618.

Mead, J.A.R., Smith, J.N., and Williams, R.T. (1955) Studies in detoxi- fication 67. The biosynthesis of the glucuronides of umbelliferone and 4-methyl-umbelliferone and their use in fluorimetric determi- nation of 8-glucuronidase. Biochem. J., 6It569-574.


Monsigny, M., Kieda, C., and Rcche, A.C. (1983) Membrane glycoproteins glycolipids and membrane lectins as recognition signals in normal and malignant cells. Biol. Cell., 47:95-110.

Musson, R.A., and Henson, P.M. (1979) Humoral and formed elements of blood modulate the response of peripheral blood monocytes. I plasma and serum inhibit and platelets enhance monocyte adherence. J . Immunol., I22:2026-2031.

Paigen, K., and Peterson, J. (1978) Coordinacy of lysosomal enzyme excretion in human urine. J. Clin. Invest., 61:751-762.

Parish, C.R. (1977) Simple model for self-non-self discrimination in invertebrates. Nature, 267:711-713.

Parish, C.R., and McKenzie, I.F.C. (1978) A sensitive rosetting method for detecting subpopulations of lymphocytes which react with allo- antisera. J . Immunol. Methods, 20:173-183.

Patt, L.M., and Grimes, W.J. (1974) Cell surface glycolipid and glyco- protein glycosyltransferases of normal and transformed cells. J. Biol. Chem., 249:4157-4165.

Payne, C.M. (1981) Platelet satellitism. An ultrastructural study. Am. J. Pathol. 203:116-128.

Perussia, B., Jankiewicz, J., and Trinchieri, G. (1982) Binding of platelets to human monocytes: A source of artifacts in the study of the specificity of antileukocyte antibodies. J. Immunol. Methods, 50:269- 276.

Rauvala, H., Prieels, J.P., and Finne, J. (1983) Cell adhesion mediated

by a purified fucosyltransferase. Proc. Natl. Acad. Sci. USA, 80:3991- 3995.

Rothenberg, B.E. (1978) The self recognition concept: An active function for the molecules of the major histocompatibility complex based on the complementary interaction of protein and carbohydrate. Dev. Comp. Immunol., 2:23-38.

Shur, B.D., and Roth, S. (1975) Cell surface glycosyltransferases. Biochim. Biophys. Acta, 415:473-512.

Turco, S.J., and Heath, E.C. (1976) The molecular and catalytic properties of galactosyltransferase from fetal calf serum. Arch. Biochem. Biophys., 176:352-357.

Vadas, M.A., David, J.R., Butterworth, A., Pisani, N.T., and Siongok, T.A. (1979) A new method for the purification of human eosinophils and neutrophils, and a comparison of the ability of these cells to damage schistosomula of schistosomu Munsoni. J. Immunol., 122: 1228-1236.

Weiser, M., and Wilson, J.R. (1981) Serum levels of glycosyltransferases and related glycoproteins as indicators of cancer. Biological and clinical implications. CRC Crit. Rev. Clin. Lab. Sci., 14t189-239.

White, R., Janoff, A,, Gordon, R., and Campbell, E. (1982) Evidence for in viuo internalisation of human leukocyte elastase by alveolar macrophages. Am. Rev. Respir. Dis., 125779-781

Winterburn, P.J., and Phelps, C.F. (1972) The significance of glycosy- lated proteins. Nature, 236:147-151.

release of galactosyltransferase from peritoneal macrophages during acute inflammation

Documents