with 1% paraformaldehyde in medium (100 ,l per well) for 15 min at room temperature, washed three times, and incubated in 200 Al of medium at 370C in a 5% ...
Proc. Natl. Acad. Sci. USA Vol. 82, pp. 1204-1208, February 1985 Immunology
Identification of a membrane-associated interleukin 1 in macrophages (T-cell proliferation/lymphokine/T-cel activation)
EVELYN A. KURT-JONES*, DAVID I. BELLER*, STEVEN B. MIZELt, AND EMIL R. UNANUE* *Department of Pathology, Harvard Medical School, Boston, MA 02115; and University, University Park, PA 16802
tMicrobiology Program, Department of Microbiology, Pennsylvania State
Communicated by Paul E. Lacy, October 9, 1984
We have found a surface membrane-associABSTRACT ated interleukin 1 (IL-i) with potent thymocyte and T-cell stimulatory activity on peptone-elicited peritoneal macrophages. The IL-1 activity was demonstrated on both fixed macrophage monolayers and on isolated membranes from unfixed macrophages. Membrane IL-1 was induced by adherence and/or by adding heat-killed Listeria monocytogenes to macrophage cultures. The macrophage membrane IL-1 was similar functionally and antigenically to soluble IL-1, but its expression could be temporally dissociated from IL-1 secretion; membrane IL-} was induced earlier and persisted longer than IL-1 secretion during in vitro macrophage culture. Moreover, when cultured macrophages that had ceased both secre, tion and membrane expression of IL4 were restimulated by adding heat-killed Listeria, substantial membrane IL-1 was indiced in the absence of detectable IL-I secretion. Membrane IL-1 appears to be an integral membrane protein since it was solubilized by detergent but was not eluted by EDTA, high salt, or low pH treatment of the membranes.
cells were maintained in medium consisting of RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 5 mM Hepes, 0.075% sodium bicarbonate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 100 ptg of penicillin per ml, 100 Ag of streptomycin per ml, 50 AtM 2-mercaptoethanol, and 5 Ag of indomethacin per ml. The viable T cells were used in proliferation assays after passage on peritoneal exudate cells (PEC) and antigen (Listeria T cells) or spleen cells and antigen (DlO.G4.1). Listeria T-cell proliferation assays were done as before (4) by culturing 4 x i04 Listeria T cells in microtiter wells (Costar 3596) containing 104 to 105 peptone-elicited macrophages per well. The macrophages were incubated with 106 heatkilled Listeria monocytogenes for 2 hr at 370C, to allow for antigen processing (8) and were fixed with paraformaldehyde, as described below, prior to the addition of the T cells. The cultures were incubated at 370C in a 5% CO2 in air atmosphere, and each well received 0.2 ACi (1 Ci = 37 GBq) of [3H]thymidine after 48 hr. The cells were harvested 18-24 hr later, and thymidine incorporation was measured by liquid scintillation spectroscopy. Soluble IL-1 Standards and Anti-IL-i Antibodies. Preparations enriched for soluble IL-1 were obtained by cycloheximide superinduction of peptone-elicited A/J macrophages, as described (9); the supernatants were collected, concentrated, and passed through a Sephadex G-75 column (Pharmacia); the soluble IL-1 fractionated with an apparent size of about 15,000 daltons. P388.D1 IL-1 was purified on an immunoaffinity column using goat anti-mouse IL-1 antibodies (10). The anti-IL-1 antibodies were produced and characterized as described elsewhere (10). Macrophage Cultures. Peritoneal macrophages were obtained 3 days after an intraperitoneal injection of 1.5 ml of 10% proteose peptone (Difco) and cultured at 370C in a 5% CO2 in air atmosphere in 96-well tissue culture plates (Costar 3596). In some experiments, 106 heat-killed Listeria monocytogenes were added per well. Fixation of Macrophages. Culture wells containing 5 x 103 to i05 macrophages per well were washed three times, fixed with 1% paraformaldehyde in medium (100 ,l per well) for 15 min at room temperature, washed three times, and incubated in 200 Al of medium at 370C in a 5% CO2 in air atmosphere usually for 24 hr. Following the 24-hr post-fixation incubation, the macrophages were washed and assayed for IL-1 activity. Preparation of Macrophage Membranes. Macrophage membranes were prepared by three methods. (i) Macrophages cultured in 24-well tissue culture plates (Costar 3424) were collected with a rubber policeman and sonicated (two 10-sec pulses with a microprobe, Fisher model 300 sonic dismembranator set at 35% power). The sonicates were centrifuged at 100,000 x g for 20 min in a Beckman Airfuge, and the
Interleukin 1 (IL-1) is a small polypeptide secreted by mononuclear phagocytes that is capable of stimulating a variety of cell types (reviewed in refs. 1-3). A prominent role of IL-1 is apparent in the regulation of T-cell growth. IL-1 maintains the spontaneous growth of thymocytes in culture and stimulates the growth of thymocytes or mature T lymphocytes induced by lectins as well as the antigen-driven proliferation of mature T cells. Indeed, it is accepted that the accessory cell function of macrophages, or other cells, not only involves the presentation of antigen in the context of the Ia glycoproteins but also the release of IL-1. IL-1 may stimulate T cells by inducing the release of interleukin 2, the T-cell growth factor, and/or the expression of interleukin 2 receptors. In this paper, we describe an IL-1 activity associated with the macrophage surface membrane that is functionally and antigenically similar to secreted IL-1. Membrane IL-1 was induced by adherence to plastic or phagocytosis of heat-killed bacteria, and its expression could be temporally dissociated from secretion of soluble IL-1. MATERIALS AND METHODS Mice. A/J, AKR/J, and C31I/HeJ mice were obtained from The Jackson Laboratory and were used at 2-6 months of age. T-Cell Lines and Clones. Listeria-specific A/J T-cell lines and clones were maintained as described (4). D10.G4.1, a conalbumin-specific AKR T-cell clone, isolated and characterized by Kaye and co-workers (5-7), was the kind gift of Charles A. Janeway, Jr. (Yale University Medical School) and was maintained by weekly passage with mitomycin Ctreated syngeneic spleen cells and conalbumin (Sigma). All T The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: IL-1, interleukin 1; PHA, phytohemagglutinin; PEC, peritoneal exudate cells.
1204
Immunology:
Kurt-Jones
et
aL
pelleted membranes were washed and assayed for IL-i activity. (ii) Macrophage membranes were prepared by hypotonic lysis. Macrophages cultured in tissue culture dishes were incubated with 10 mM Tris HCl (pH 7.4) and allowed to swell for 5 min on ice. An aliquot of 60% sucrose in 5 mM Tris HCl was added to restore isotonicity (final concentration of 10% sucrose). The macrophages were immediately collected with a rubber policeman and disrupted by 10 strokes with a tight-fitting Dounce pestle. Nuclei and cell debris were removed by centrifugation at 500 x g for 20 min. The cell membranes were pelleted by centrifugation at 48,000 x g for 20 min, washed twice with 100 nM NaCl/50 mM Tris'HCl, pH 7.4, and assayed for IL-1 activity. (iii) Membranes were further fractionated by using self-forming Percoll gradients (11). Briefly, membrane preparations obtained by hypotonic lysis were suspended in 27% Percoll/0.25 M sucrose/10 mM EDTA and centrifuged at 28,000 x g for 60 min in a Sorvall SS34 rotor. An aliquot of live macrophages was labeled with 1251 by the lactoperoxidase method (12) to follow separation of the plasma membranes. The plasma membranes were found in a single band with an average density of 1.032 g/ml. However, evaluation of the distribution of 83-glucuronidase and N-acetylglucosaminidase (13) revealed the presence, as well, of intracellular membranes. Macrophage membranes, isolated by hypotonic lysis, were detergent extracted by resuspending the washed 48,000 x g pellet in 60 mM N-octyl glucoside (Sigma) and incubating for 15-60 min at room temperature. Detergent-insoluble material was pelleted by centrifugation and washed. The detergent-solubilized material was dialyzed extensively against 100 mM NaCl/50 mM Tris-HCl, pH 7.4, in dialysis tubing with a 6000- to 8000-dalton cutoff (Spectrapor no. 1, Spectrum Medical Industries, Los Angeles). IL-1 Assays. IL-1 activity was measured in three separate assays; serial dilutions of IL-1-containing material were cultured in 200 tkl of medium in flat-bottom tissue culture wells (Costar 3596) with (i) 106 A/J or C3H/HeJ thymocytes, (ii) 106 thymocytes and 5 ,ug of phytohemagglutinin (PHA) (Difco) per ml, and (iii) 2 x 104 DIO.G4.1 T cells and 2.5 ,ug of concanavalin A (Con A, Sigma) per ml. All cultures were incubated at 37°C in a 5% CO2 in air atmosphere. After 48 hr, each well was pulsed with 0.2 ,uCi of [3H]thymidine and then harvested 18-24 hr later. All determinations were done in triplicate and are shown as the mean cpm SD of [3H]thymidine incorporation in response to representative dilutions of fixed macrophages and soluble IL-1-containing supernates or in units of IL-1. One unit of IL-1 per milliliter was defined as the concentration required to produce half-maximal proliferation of thymocytes or D10.G4.1 cells. The soluble IL-1 standards used throughout these experiments contained 20-40 units of IL-1 per ml when assayed with thymocytes, with or without PHA, and 50-150 units of IL-1 per ml when assayed with D10.G4.1 plus Con A. The data are presented as thymocyte units/ml or D10.G4.1 units/ml to indicate the cell type used to titrate the soluble IL-1 standard in that experiment. RESULTS Stimulation of T-Cell Lines and Thymocytes by Fixed Macrophages. A major impetus for this study came from our demonstration that Listeria-specific T-cell lines proliferated in an I-region-restricted, antigen-specific manner to peritoneal macrophages pulsed with antigen and subsequently fixed in paraformaldehyde. The T-cell response to these fixed macrophages was independent of the secretion of IL-1, since the fixed macrophages were metabolically inert, and was not affected by the addition of IL-i-containing supernatants to the proliferation assay (4). These experiments,
Proc. NatL. Acad. Sci. USA 82 (1985)
1205
therefore, questioned the role of IL-1 in the regulation of the growth of activated T cells. The present studies represent an evaluation of the stimulatory activities of these fixed macrophages. Macrophages were harvested, planted in culture dishes, usually with heat-killed Listeria organisms as an added stimTable 1. Paraformaldehyde-fixed macrophages have cell-associated IL-1
[VH]Thymidine incorporation, cpm Listeria T Thymocytes (PHA)a
cellsb
Experiment 1 Listeria-pulsed, fixed 13,560 ± 3385 (-) macrophagesc Supernate from fixed macrophages [24-84 hr after fixation; 50% 78 ± 25 (-) (vol/vol)] Experiment 2 5,091 + 1332 (-) 7,082 + 238 Listeria-pulsed, fixed 10,105 ± 1568 (+) macrophages 188 ± 38 Unpulsed, fixed 1,983 ± 336 (-) 7,126 + 854 (+) macrophages Experiment 3 Listeria-pulsed, fixed macrophages No addition 9,491 ± 1452 (+) 1,494 ± 535 (+) Anti-IL-1, 10 jug/ml 7,528 + 550 (+) Control Ig, 10 jg/ml 1,353 ± 128 (±) Anti-IL-1, 40 jug/ml Control Ig, 40 jug/ml 8,463 ± 418 (+) Soluble IL-1, 1 unit/ml 3,522 + 830 (+) No addition 2,388 t 608 ( -) Anti-IL-1, 10 jug/ml 4,297 + 57 (+) Control Ig. 10 ,ug/ml 2.055 + 297 (+) Anti-IL-1, 40 j.tg/ml Control Ig. 40 ,ug/ml 3,787 + 816 (+) Experiment 4d Listeria-pulsed, fixed macrophages 13.459 ± 2864 (+) 29,626 + 1874 No addition Control Ig. 25 ug/ml 11,027 ± 948 (+) 31,172 + 2993 6.864 + 874 (-) 12,236 ± 646 Anti-IL-1, 5 jug/ml 1,926 + 211 (+) 1,283 + 83 Anti-IL-1, 25 ,ug/ml +2 units of IL-1/mle 5,576 + 1041 (+) 9,392 ± 728 +-4 units of IL-1/ml 9,440 ± 508 (+) 21,644 + 1483 12.018 + 2247 ( +) 29,148 ± 3621 Anti-Mac-1, 5 jug/ml 9,834 ± 693 (+) 28,942 ± 1292 Anti-Mac-1, 25 ug/ml Data are shown as mean cpm + SD of [3H]thymidine incorporation of triplicate cultures incubated for 48 hr at 37C. pulsed, and harvested 18-24 hr later. 'A/J thymocytes (106 per well) with (+) or without (-) 5 jug of PHA per ml in 200 ,ul. Background [3H]thymidine incorporation was 400-600 cpm without PHA and 900-1200 cpm with PHA. 'Listeria T cells (4 x 104 per well) in 200 ,ul. Background [3H]thymidine incorporation was 50-110 cpm. cMacrophages were cultured at 104 to 105 per well with 106 heatkilled Listeria per well for 1-1i/2 hr at 37°C. Macrophages were washed, fixed, and processed. The response of thymocytes and D1O.G4.1 cells on 8 x 104 to 105 macrophages per well is shown. The supernate was assayed at 25-50%. The response to 50% supernate is shown. dGoat anti-IL-1 and control goat anti-rabbit Ig antibodies were prepared by ammonium sulfate precipitation (10). Anti-Mac-1 antibodies were obtained from culture supernates of hybridoma
Ma/70.1.
I1
secreted IL-1. containing 40 units of -I per ml for thymocytes plus PHA. Not shown are virtually identical results obtained by using the purified P388D.1 IL-1.
eCycloheximide-induced
1206
Immunology: Kurt-Jones et
Proc. NatL Acad Sci. USA 82
al.
ulus, and then fixed. (The fixed cells were always tested 24 hr or more after fixation at a time when no soluble IL-1 was released from the cells.) These fixed macrophages stimulated the proliferation of thymocytes from normal mice in the absence or presence of PHA (Table 1, experiments 1 and 2). This stimulatory activity, which we have termed "membrane IL-1," was found in the absence of IL-1 released from the fixed cells (Table 1, experiment 1). As noted in experiment 2 of Table 1, the fixed macrophages not pulsed with Listeria also contained the membrane IL-1 activity, although at lower levels than the Listeria-pulsed cells. As noted before, the Listeria-pulsed fixed macrophages also supported the proliferation of Listeria-specific T-cell lines. A goat anti-IL-1 blocked membrane IL-1 activity, as well as soluble IL-i, in a dose-dependent manner (Table 1, experiments 3 and 4). The anti-IL-1 inhibition of thymocyte proliferation to membrane IL-1 could be overcome by the addition of soluble IL-1 (Table 1, experiment 4). Similarly, Listeria T-cell proliferation to Listeria-pulsed, fixed macrophages was abrogated by the anti-IL-1 and could be reconstituted with soluble IL-1 (Table 1, experiment 4). Antibodies to another macrophage membrane protein, Mac-1, did not block either thymocyte or T-cell responses. Recently, Kaye and co-workers (5-7) have studied a conalbumin-specific, AKR T-cell clone, DlO.G4.1, that proliferates in a dose-dependent manner to soluble IL-1 in the presence of Con A. This line can therefore be used to detect and titrate IL-1 by adding the test materials together with Con A. Fig. 1 shows that D1O.G4.1 plus Con A cells also proliferated in response to fixed macrophages. In the absence of Con A, D1O.G4.1 cells did not proliferate to any concentration of fixed macrophages or soluble IL-1. Membrane IL-1 activity for thymocytes and D1O.G4.1 was found on Ia-negative as well as Ia-positive macrophages. Peptone exudates from A/J mice, which have low numbers of Iak-positive macrophages, stimulated thymocytes even after treatment with anti-I-Ak and complement (in two experiments, the thymocyte response was 96-103% of the control response, and the D1O.G4.1 response was 94-112% of control). On the other hand, antigen presentation to Listeria T cells was abrogated by this treatment to the extent of 89100% inhibition of proliferation. Regulation of Membrane and Secreted IL-1. Freshly isolated peritoneal macrophages had no detectable membrane IL1 activity upon fixation, and, if such macrophages were kept in suspension for up to 2 hr at 37°C, there was low or undetectable IL-1 activity (Table 2). Membrane IL-1 activity, however, was induced by adding heat-killed Listeria organisms to the suspension of macrophages. More strikingly, adherence of the macrophage to the tissue culture wells stimulated strong membrane IL-1 activity (Table 2, experiments 1 and 2). Adherence has been shown to trigger IL-1 secretion (ref. 9 and Fig. 4) as well as the ability of macrophages to express Ia in response to a T-cell lymphokine (14). Macrophages planted without added stimulus showed membrane IL-1 activity at 24 hr, declining to very low levels by the third and fourth day in culture (Fig. 2). During this 4day period, the amount of secreted IL-1 was very low. Macrophages planted with addition of heat-killed Listeria during the first 24 hr maintained their membrane IL-1 for the 4 days; soluble IL-1, however, was released mainly during the first 24-hr period (Fig. 2). Fig. 3 shows that the appearance of membrane IL-1 in this latter group also preceded the release of soluble IL-1. Membrane IL-1 was induced in the absence of secreted IL-1 by delaying the addition of Listeria for 2 or more days after the initiation of the cultures (Fig. 4). Nature of Membrane IL-1. Membranes prepared from Listeria-pulsed, unfixed macrophages showed significant IL-1 activity when assayed on either thymocytes or D1O.G4.1 cells
(Table 3).
The membranes that we tested were
(i) crude
(1985)
X40
4
x
0. u
o
C)
30
0. 0 u
20
._
._ 10
C
5x 10'
104
5 X 104
i05
PEC, no. per well
FIG. 1. Fixed PEC stimulate thymocyte and DiO.04.1 proliferation. Peptone-elicited PEC were plated in 96-well culture plates (Costar 3596) with 106 heat-killed L. monocytogenes per well. After a 3-hr incubation at 370C, the macrophage monolayers were washed and fixed. Twenty-four hours after fixation, macrophages were washed and 2 x 104 D10.G4.1 T cells plus 2.5 Ag of Con A per ml (o) or 106 A/J thymocytes plus S Ag of PHA per ml (U) were added to each well. The cultures were pulsed with 0.2 ,uCi of [3H]thymidine per well during the last 24 hr of a 72-hr incubation at 370C. The data shown are the mean cpm ± SD of thymidine incorporation in triplicate wells. The extent of [3H]thymidine incorporation by D1O.G4.1 cells in the absence of fixed macrophages was medium, 90 ± 9; Con A, 109 ± 21; 10% (vol/vol) IL-1 standard, 260 ± 14; 10% IL-1 standard plus Con A, 54,057 ± 3443. Thymidine incorporation by thymocytes in the absence of fixed macrophages was medium, 151 ± 1; PHA, 1101 ± 213; 10% IL-1 standard, 4501 ± 1217; 10% IL-1 standard plus PHA, 16,806 ± 2299. Table 2. Macrophage membrane IL-1 is induced by adherence and phagocytosis
[3H]Thymidine Macrophage incubation" Unpulsed Fixed in suspension Fixed after adherence
Listeria-pulsed Fixed in suspension Fixed after adherence
incorporation, cpm Time of fixation, Thymocytes D1O.G4.1 + hr + PHAb Con AC 0 1 2 1 2
0 1 2 1 2
261 ± 34 642 ± 67
3885
± 310
561 ± 131 2088 ± 342 6886 ± 515
542 106 430 33,122 33,560
± 84 ± 75 ± 175 ± 2689 ± 2811
827 3,963 24,891 33,130 38,530
± 439 ± 1718 ± 2274 ± 1764
± 245
Data are shown as mean cpm ± SD of [3H]thymidine incorporation of triplicate cultures. aMacrophages, at 1-5 x 106 per ml, were incubated with 10 heat-killed Listeria per macrophage at 370C for the indicated times, fixed, and processed. Macrophages were kept in suspension in polypropylene tubes (5 ml per tube) by swirling every 10 min or were adhered by stationary culture of 100 Atl of the cell suspension in 96-well tissue culture plates (Costar 3596). A single macrophage preparation was used for each experiment. bA/J thymocytes (106 per well) plus 5 Ag of PHA per ml in 200 Al. Background [3HIthymidine incorporation was 327 ± 25 cpm. CDlO.G4.1 (2 x 104 cells per well) plus 2.5 jug of Con A per ml in 200 Al. Background [3H]thymidine incorporation was 1072 ± 141 cpm.
NatL AcaL Sci. USA 82 (1985) Kurt-Jones ~~Proc. etet aLaL Immunology: Kurt-Jones Immunology:
1207
0l x
*44j~~~ (32) ~~(20)
4030-
.~20-(03)
2
lo(0.6)
30 60
120
180
Time in culture, min 12
3
4
FIG. 3. Membrane IL-i appears earlier than soluble IL-i. PEC Listeria, fixed, and assayed as described in the legend to Fig. 2. The D1O.G4.1 plus Con A response to i0' macrophages per well (m) and supernates from the same macrophages assayed at 50% concentration is shown. m, Membrane IL-i, Listeria-pulsed PEC; n, secreted IL-i, Listeria-pulsed PEC.
Day of culture FIG. 2. Membrane IL-i persists longer than IL-i secretion in culture. PEC were plated at iO' to 1W cells per well and incubated with (in, w) or without (o, 0) 106 heat-killed Listeria per well for the first 24 hours. Supernates were collected, and the macrophage monolayers were washed at 24-hr intervals and fixed. Titrations of supemnates (Ei, o) and fixed macrophages (in, e) were assayed for IL1 activity by adding 2 x 104 Dl0.G4.1 T cells per well with 2.5 Ag of Con A per ml. This graph shows the D10.G4.1 plus Con A response to 104 macrophages per well and supernates from the same macrophages assayed at 25% final concentration and is representative of the response at higher and lower concentrations of fixed macrophages and supernates. The units of IL-i per ml are shown in parentheses and were determined by comparison with the response of DiO.G4.1 cells to a soluble IL-i standard titrated from 0.01 to 20 units/mI. u, Membrane IL-i, Listeria-pulsed PEC; m, secreted IL-1, Listeria-pulsed PEC;eo, membrane IL-i, unpulsed PEC; 0, secreted IL-i, unpulsed PEC.
were incubated with heat-killed
detergent (60 mM N-octyl glucoside; Table 3, experiment 3). In the same experiment, treatment with iO mM EDTA or i M sodium chloride or by incubation at pH 3 released
