Pro-interleukin-1β production by a subpopulation of human T cells, but not NK cells, in response to interleukin-2

CELLULAR

IMMUNOLOGY

130,118-128

(1990)

Pro-lnterleukin-l fl Production by a Subpopulation of Human T Cells, but not NK Ceils, in Response to Interleukin-2 ROBERT

P. NUMEROF,’

ALAN AND

N. KOTIK, JAMES

W.

CHARLES

A. DINARELLO,

MIER

Immunology Program, Sackler School of Graduate Biomedical Sciences, Tufts University: and the Department ofMedicine, New England Medical Center, 750 Washington Street, Boston, Massachusetts 02111 Received February 27, 1990; accepted April 27, 1990 Previous studies have shown that IL-2-stimulated (PBMC)

produce

IL-I/3

and TNFa

and that monocytes

peripheral

blood mononuclear

are the primary

source

cells

of these IL-2-

inducible cytokines. In this report, we provide evidence that monocytes are not the only source. We examined cytokine production by IL-2-treated, nonmonocytic PBMC and found that a population of nonadherent low-density cells (NLDC) produced both IL-10 and TNFa in response to IL-2. IL-l@ was synthesized by IL-2-treated NLDC as the 35kDa intracellular precursor (pro-IL- l@), but was neither secreted nor processed to the mature 17-kDa form of the molecule. To determine which cells within the NLDC population generated pro-IL-lp and TNFo( in response to IL-2, we positively selected NK cells and T cells, the two major components of NLDC, using specific monoclonal antibodies. Although IL-2-treated CD 16+ NK cells produced TNFol and transcribed IL-l/3 mRNA, they did not synthesize the IL-10 protein. Conversely, LPS-treated CD 16+ and IL-2-treated CD4+ and CD5+ NLDC produced elevated levels of both TNFol and IL- 10. Our findings illustrate the complex nature of IL- 10 production by IL-2-stimulated PBMC and suggest that the factors controlling IL- I fl gene transcription and translation, as well as secretion and processing, vary widely as a function of cell type and stimulus. o 1990 Academic

Press, Inc.

INTRODUCTION IL- 1a and IL- 1p are produced by a large variety of cell types in response to a wide array of stimuli (1,2). Human peripheral blood mononuclear cells (PBMC)* produce both forms of IL- 1 in response to IL-2 in vitro (3). Although monocytes are the main source of IL- 1 within this mixture of cells, several additional PBMC subpopulations could be contributors to the overall production. The ability of NK cells and T cells to respond to high concentrations of IL-2 by secreting IFN-7 and developing lympho’ To whom correspondence and reprint requests should be addressed at New England Medical Center, Box 245,750 Washington St., Boston, MA 02 I I 1. ’ Abbreviations used: PBMC, peripheral blood mononuclear cells; NLDC, nonadherent lowdensity cells; LAK, lymphokine-activated killer; LPS, lipopolysaccharide; rIL-2, recombinant interleukin-2; proIL-l 0, interleukin- 10 precursor; RIA, radioimmunoassay; FACS, fluorescence-activated cell sorter; FcRIII, Fc-y receptor (CD I6 antigen). 118 0008-8749190 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved

IL-2

STIMULATES

PRO-IL-l!3

SYNTHESIS

IN

NLDC

119

kine-activated killer (LAK) activity has been well described (4-8). Furthermore, human NK cells and a murine T cell clone have been shown to produce IL- 1 in response to LPS and antigen, respectively (9, 10). These observations suggest that NK cells and T cells may be capable of producing IL- 1 in response to IL-2. The major extracellular species of IL- 1 produced by IL-2-stimulated PBMC is ILl/3 (3), and IL-lp also predominates at the level of mRNA expression (11). We therefore decided to focus our attention on IL-lb in our investigation of nonmonocytic cell sources of IL-2-induced IL- 1 production. IL-10 and TNFa are functionally related cytokines (I), which are generally produced by PBMC in vitro in response to the same inducing agents (12). Like IL-ID, TNFa is produced by PBMC following IL-2 stimulation (13). However, the cells within the PBMC mixture which participate in the production of these two cytokines might differ. Thus we compared the abilities of different PBMC subpopulations to produce not only IL- I@, but also TNFa, in response to IL-2. MATERIALS

AND METHODS

Reagents. Escherichia coli lipopolysaccharide (LPS) was purchased from Sigma Chemical Co. (St. Louis, MO). Human recombinant IL-2 (rIL-2), produced in E. coli and purified to homogeneity, was generously provided by Cetus Corp. (Emeryville, CA). The rIL-2 preparations (3 X 1O6 Cetus Units/mg; 18 X 1O6 IU/mg) contained less than 0.0 1 ng of endotoxin/mg protein as determined by the Limulus assay. Human rIL- 1p and rTNFa, also produced in E. coli and purified to homogeneity, were provided by Cistron Biotechnology (Pine Brook, NJ) and Genentech (South San Francisco, CA), respectively. ‘2sI-labeled cytokines were prepared as described previously (12, 14). Preparation of PBMC and PBMC subpopulations. PBMC were obtained from the heparinized venous blood of healthy normal volunteers by density gradient centrifugation with Lymphocyte Separation Medium (Organon Teknika Corporation, Durham, NC). Monocyte-depleted PBMC were prepared by incubating PBMC in plastic flasks for 90 min at 37°C. The plastic-nonadherent cells were recovered and passed over nylon wool columns (Cellular Products Inc., Buffalo, NY) to remove any residual monocytes and most B cells (15). To separate the plastic- and nylon wool-nonadherent cells according to cell density, Percoll discontinuous gradient centrifugation was employed ( 16). Briefly, the osmolality of Percoll (Pharmacia, Uppsala, Sweden) was adjusted to 290 mOsm/kg H20 with 10X concentrated Dulbecco’s PBS (GIBCO, Grand Island, NY). Seven fractions of Percoll in medium, ranging from 40.8 to 66.6% Percoll, were successively layered into 15-ml plastic conical test tubes and overlaid with 6-7 X lo6 nonadherent cells. Following centrifugation, cells from each interface were removed with a Pasteur pipet and washed. The low-density cells collected from the interfaces between fractions 1 and 2 and fractions 2 and 3 were combined to form the nonadherent low density cell (NLDC) population. In some experiments the NLDC were stained sequentially with a murine anti-human monoclonal antibody and fluoresceinated goat F(ab’), anti-mouse IgG and IgM (Organon Teknika-Cappel, Durham, NC), and sorted by flow cytometry with an Epics 541 FACS (Coulter Electronics Inc., Hialeah, FL). Alternatively, cells were incubated with a monoclonal antibody and antibody-positive cells were separated from antibody-negative cells by using Dynabeads coated with sheep anti-mouse IgG (Dynal Inc., Great Neck, NY).

120

NUMEROF

ET AL.

Phenotyping of cells. Cell surface phenotype was determined by indirect immunofluorescence and flow cytometry (Epics 541 FACS). 5 X lo5 cells were stained with a mAb at the dilution recommended by the manufacturer, washed in FACS buffer (PBS, 0.1% BSA, 0.1% NaN,), restained with fluoresceinated goat F(ab’)Z anti-mouse IgG and IgM (Organon Teknika-Cappel), and then washed again. The mAb antiLeu-1 (anti-CDS, T cells), anti-Leu-3a (antiCD4, T helpers), anti-Leu-1 lb (antiCD 16, NK cells), anti-Leu- 16 (antiCD20, B cells), anti-Leu-M 1 (anti-CD 15, monocytes/granulocytes), and anti-Leu-M3 (anti-CD 14, monocytes) were obtained from Becton-Dickinson (Mountain View, CA) and the mAb OKT3 (anti-CD3, T cells) was obtained from Ortho Diagnostics (Raritan, NJ). Cell culture and RIA. RPM1 1640 medium (GIBCO) supplemented with L-glutamine (2 m&Q, penicillin ( 100 U/ml), streptomycin ( 100 @g/ml), Hepes ( 1 mM), and either 5 or 10% heat-inactivated fetal bovine serum (HyClone Laboratories, Logan, UT) was used as culture medium in all experiments. For cytokine production experiments, cells were incubated in duplicate in polypropylene tubes at a density of 5 X lo6 cells/ml in medium alone or medium containing LPS or rIL-2. With the exception of LPS-containing medium, all culture media contained 5 pgfml polymyxin B (Pfizer Inc., New York, NY), an endotoxin inhibitor. In experiments measuring the total (secreted plus cell-associated) production of inducible cytokines, the cell suspensions were frozen and thawed three times in the presence of 1 mMPMSF (Sigma Chemical Co., St. Louis, MO). In other experiments, supematants and cells were separated by centrifugation, and the cells were washed and resuspended in a volume of fresh medium equal to that of the supernatant prior to freezing and thawing. The concentrations of IL-l/3 and TNFa in the various cell lysates and culture supematants were determined by using radioimmunoassays (RIA) specific for each cytokine ( 12, 14). Briefly, 100 ~1 of the samples or sample dilutions were incubated for 24 hr at room temperature with 100 ~1 of the appropriate anti-cytokine antiserum diluted in RIA buffer (PBS, 0.25% BSA, 0.1% NaNx) and 300 ~1 of RIA buffer containing 0.3% normal rabbit serum. RIA buffer (100 ~1) containing 10,000 cpm of 1251labeled recombinant cytokine was then added and the mixtures were incubated for an additional 24 hr. Finally, a total of 500 ~1 of RIA buffer containing sheep antirabbit IgG antiserum (2%) and polyethylene glycol(7%) was added, the mixture was centrifuged, and the radioactivity of the precipitate was determined with a gamma counter. The concentrations of IL-l@ and TNFa in the samples were determined from standard curves generated from known concentrations of recombinant cytokines. The threshold of detectability for the two cytokines was 0.1 rig/ml. All samples were assayed in duplicate and SEM were consistently less than 6% of the measured values. Radiolabeling and immunoprecipitation. NLDC were cultured in methionine-free RPM1 1640 medium (Flow Laboratories, McLean, VA) supplemented with 2 mM L-glutamine, 3% PBS-dialyzed fetal bovine serum, and 100 &i/ml [35S]methionine (800 Ci/mmol, New England Nuclear, Boston, MA), along with LPS or rIL-2, for 24 hr at 37°C. Supematants and cells were separated by centrifugation and the cells were lysed in buffer containing 10 mM Na2HP04, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 1 mM PMSF, and 1% NP-40 ( 17). Supernatants and cell lysates were precleared twice with 1% normal rabbit serum and 10% protein A-agarose (Genzyme, Boston, MA). Specific immunoprecipitation was subsequently performed with antisera raised against rIL- 1P ( 18) and 10% protein A-agarose. Immuno-

IL-2 STIMULATES

PRO-IL-l@ SYNTHESIS

IN NLDC

121

precipitates were washed sequentially in wash buffer (10 mM Na2HP04, pH 7.4, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 1 mM PMSF, 1% NP-40, and 0.5% sodium deoxycholate), wash buffer containing 0.05% SDS, and wash buffer containing 0.5 h4 NaCl (17). Immunoprecipitates were then resuspended in Laemmli sample buffer, boiled for 5 min and analyzed by SDS-PAGE (13% acrylamide) and fluorography ( 19). Chromatofocusing, Polybuffer exchanger 94 (Pharmacia, Uppsala, Sweden) was equilibrated with 25 Mimidazole-HCl, pH 7.4, and packed in a column. Lysates of IL-2-stimulated NLDC were applied to the column and 1 ml fractions were collected during elution with polybuffer 74-HCl, pH 4.0 (Pharmacia). The pH and IL-l@ content of each fraction were determined using a pH meter (Amber Science, San Diego, CA) and the IL- l/3 RIA, respectively. RNA isolation and Northern analysis. Total cellular RNA was isolated from CD 16+ cells by the GITC/CsCl method (20). The RNA from each sample was denatured by glyoxalation and separated on a 1.0% agarose gel (21). Ethidium bromide staining demonstrated that equivalent amounts of RNA were applied to each lane of the gel. The RNA was transferred to Genescreen Plus (New England Nuclear, Boston, MA) by capillary blotting in 5X SSC. Filters were prehybridized for 2 hr at 42°C in 50% formamide, 5X SET, 1X Denhardt’s solution, 1% SDS, 50 m&f Na2HP04, pH 6.6, and 100 pg/ml sonicated salmon testes DNA. Hybridization was carried out for 16 hr at 42°C in 50% formamide, 5X SET, 1X Denhardt’s solution, 1% SDS, 20 mM Na2HP04, pH 6.6, 10% dextran sulfate, 25 pg/ml sonicated salmon testes DNA, and 5 X lo5 cpm/ml radiolabeled cDNA probe. Filters were then washed to a stringency of 1X SET, 0.1% SDS at 50°C and autoradiographed at -80°C with Kodak XAR-5 film and intensifying screens (New England Nuclear). The cDNA probe for human IL- lp was isolated from pGEM-IL- 1p by restriction endonuclease digestion and electroelution (22). The insert was labeled by the random primer method (BoeringherMannheim, Indianapolis, IN) to a specific activity of l-2 X lo9 cpm/pg DNA with [32P]dCTP (3000 Ci/mmole; New England Nuclear). RESULTS We and others have demonstrated that monocytes respond directly to IL-2 by producing IL- ID and TNFa, and that monocytes are the primary source of these cytokines in cultures of IL-2-stimulated PBMC (3, 13,23). In order to determine whether monocytes are, in fact, the only cells within the PBMC population capable of producing IL- l/3 and TNFa in response to IL-2, we examined cytokine levels in cultures of unfractionated and monocyte-depleted PBMC, each treated with 1000 U/ml IL-2 for 24 hr. Since the IL-l produced by PBMC is detectable in both cell-associated and secreted forms (24) we combined cell lysates and supernatants to more accurately assesstotal cytokine production. As shown in Table 1, plastic and nylon wool adherence reduced the number of monocytes (CD 14+ and CD 15+ cells) present to between 0 and 1 percent of the PBMC, and the removal of monocytes coincided with a marked reduction in the amounts of IL-l@ and TNFol produced in response to IL-2. Nevertheless, IL-2-induced cytokine production did indeed occur in the monocyte-depleted PBMC cultures and approximately lo-15% of the IL-2-inducible cytokine production by PBMC can be attributed to the nonadherent cells. To characterize the nonmonocytic PBMC which contribute to IL-Zinducible cytokine production, nonadherent cells were placed on discontinuous Percoll gradients

122

NUMEROF TABLE

ET AL. 1

Effect of Monocyte Depletion on the Ability of PBMC to Produce IL- lfl and TNFa in Response to IL-2 Cytokine production b % Positive cells”

PBMC NAPBMC’

IL-lb (rig/ml)

TNFa (@ml)

CD3

CD16

CD20

CD14

CD15

0

IL-2

0

IL-2

65 70

16 20

9 3

24 0

19 1


11.0 1.4


2.2 0.3

a As determined by FACS analysis. ’ Cytokine production in whole culture lysates after 24 hr of incubation in medium alone or medium containing rIL-2 (1000 U/ml). ’ Nonadherent PBMC, i.e., cells remaining after sequential plastic and nylon wool adherence steps.

and separated according to cell density. As shown in Fig. 1, the low-density cells which migrate to the first two interfaces in the gradient (NLDC) generally produced levels of IL- 16 and TNFa in excess of 0.1 rig/ml during incubation in media alone. These baseline levels of production were augmented at least 3- to 15fold by the,addition of IL-2. The high-density cells, which are almost exclusively small, resting T cells, did not produce IL- lp and TNFa! either spontaneously or following IL-2 stimulation. To determine if the IL-lp produced by these cells exists in both cell-associated and secreted forms, we stimulated NLDC with either LPS or IL-2 in the presence of [35S]methionine, precipitated [35S]methionine-labeled IL-lp in both the cell lysates and supernatants with an anti-IL-l/3 antibody, and examined the immunoprecipitates with SDS-PAGE and fluorography. As shown in Fig. 2, LPS-treated NLDC produced both the 35-kDa intracellular and 17-kDa extracellular forms of IL-l& while the IL-2-stimulated NLDC produced only the 35kDa intracellular precursor (pro-IL-l/l). This result is consistent with the lack of contaminating monocytes in the NLDC preparations, since monocyte-enriched populations are fully capable of secreting and processing IL- 1,6in response to IL-2 (3).

FIG. 1. Characterization by density gradient centrifugation of nonadherent PBMC which produce and TNFol. Nonadherent PBMC were separated by Percoll discontinuous gradient centrifugation. collected from each interface in the gradient were incubated in medium containing 1000 U/ml rIL-2 bars) or medium alone (light bars) for 24 hr. Whole culture lysates were assayed for IL- 1s and TNFa specific RIA. Arrows indicate the threshold of detectability for each RIA.

IL-ID Cells (dark using

IL-2 STIMULATES

PRO-IL-ID SYNTHESIS 123

IN NLJX

123

456

FIG. 2. Immunoreactive intra- and extracellular IL-l@ produced by LPS- and IL-2-treated NLDC. NLDC were labeled with [35S]methionine in the presence of LPS ( 100 rig/ml) or rIL-2 (1000 U/ml). Cell lysates of LPS-treated (lanes 1 and 2) and IL-2-treated (lanes 4 and 5) NLDC were immunoprecipitated with normal rabbit serum (lanes 1 and 4) or an anti-rIL-10 antiserum (lanes 2 and 5). Supernatants of LPStreated (lane 3) and IL-2-treated (lane 6) NLDC were immunoprecipitated with the anti-rIL-lp antiserum. Immunoprecipitates were analyzed by SDS-PAGE and fluorography. The positions of the m.w. standards are indicated to the left of lane 1.

It is possible that the IL-2-induced pro-IL- 1p detected in the radioimmunoprecipitates is distinct from that resulting from LPS stimulation and that a difference in biochemical structure may be responsible for the failure of IL-Zstimulated NLDC to cleave the IL- 1p precursor and secrete the mature form of the molecule. As shown in Fig. 2, the molecular weight of IL-2-induced pro-IL- 1p is identical to that resulting from LPS stimulation. To investigate another potential biochemical difference, we determined the isoelectric point of pro-IL-l@ in IL-2-stimulated NLDC. The major peak of intracellular IL-l@ was found in the acidic pH range of 5.7-5.8 (Fig. 3) as has been observed by others for pro-IL- 1p in LPS-stimulated monocytes (25). Thus, neither the molecular weight nor the isoelectric point of the pro-IL- 1p induced by IL2 appears to differ from that induced by LPS. 1.0

r

FIG. 3. Isoelectric point determination of intracellular IL-l@ in IL-2-stimulated NLDC. Lysates of IL-2stimulated NLDC were applied to a chromatofocusing column. The pH and IL-16 content ofeach fraction eluted from the column were measured. Open circles denote fractions for which the amount of IL-10 present was below the threshold of detectability (as indicated by the dotted line) for the IL-l/3 RIA.

NUMEROF

124

ET AL.

TABLE 2 Ability of NLDC Subpopulations to Produce IL-l@ and TNFa in Response to IL-2 Cytokine production“ IL-l@ (@ml) Cells

TNFa (rig/ml)

0

IL-2

0

IL-2

%CD16+ b 35 98


1.6
0.2 0.5

1.5 1.6

Unsorted Sorted CD5+

90CD5+ 38 96

0.2 0.5

1.6 0.9

0.2 0.4

0.6 0.8

Unsorted Sorted CD4*

% CD4+ 44 92

0.9 3.1

2.7 4.4

Unsorted Sorted CD 16+

’ Cytokine concentration in whole culture lysates after 24 hr of incubation in medium alone or medium containing rIL-2 (1000 U/ml). ’ As determined by FACS analysis.

In order to determine which low density nonadherent cells are responsible for IL2-induced IL- l/3 and TNFa production, we initially sorted for CD 16+ and CDS+ cells to obtain populations enriched for NK cells and T cells, the two major components of the low-density Percoll fractions. Unsorted and positively sorted cells were each incubated in media, with or without IL-2, for 24 hr and whole culture lysates (cells plus supernatants) were assayed for IL- 1p and TNFa. Compared to unstimulated cells, IL-2-stimulated CD16+ cells produced elevated levels of TNFa, but not IL-l/3 (Table 2). In contrast, IL-2-stimulated CDS’ cells produced increased levels of both cytokines. As shown in Table 2, low-density cells were sorted for another T cell marker, CD4, and these positively selected T cells also produced increased levels of IL- l@ in response to IL-2. In order to determine if the failure of NK cells to produce IL-lp was a result of prior exposure to the anti-CD 16 antibody employed in the sorting, we treated positively selected CD16+ cells with an alternative stimulus, LPS. As shown in Table 3, TABLE 3 Comparison of IL-2 and LPS as Inducers of IL- Ifl and TNFa Production by CD 16+ Cells Cytokine production’ Stimulus 0

IL-2 LPS

IL-lP(ng/ml) 0.2 0.2 0.7

TNFa (rig/ml) 0.6 I.0 1.2

o Cytokine concentration in whole culture lysates after 24 hr of incubation in medium alone or medium containing rIL-2 (1000 U/ml) or LPS (100 t&ml).

IL-2 STIMULATES

PRO-IL-I@ SYNTHESIS

28s

-

18s

-

IN NLDC

125

FIG. 4. Accumulation of IL-10 mRNA in LPS- and IL-2-stimulated CD16+ cells. Total cellular RNA was extracted from positively selected CD16+ cells stimulated with LPS (100 rig/ml), rIL-2 (1000 U/ml), or medium alone for 4 hr. The RNA (5 &lane) was electrophoresed, transferred to Genescreen Plus, and hybridized to the 32P-labeled IL- l/3 cDNA probe. The positions of the 18s and 28s ribosomal subunits are indicated to the left of the figure.

these LPS-stimulated CD16+ cells produced IL-l@ This result is consistent with a previous report demonstrating LPS-induced IL-1 synthesis by NK cells (9). Moreover, it suggests that NK cells obtained through positive selection are not rendered unresponsive as a consequence of prior exposure to the anti-CD 16 antibody. Finally, we investigated whether IL-2 can stimulate IL-lp gene transcription in CD 16+ cells. NLDC were positively selected for the CD1 6 marker and subsequently treated with LPS, IL-2, or media alone for 4 hr. Northern analysis of total cellular RNA demonstrated that IL-2 induced an increase in the level of IL-l/3 mRNA, although this increase was less than that induced by LPS (Fig. 4). DISCUSSION We have previously shown that monocytes are the primary source of the IL-lb produced by PBMC in vitro in response to IL-2 (3). However, they are clearly not the only source since monocyte-depleted PBMC also generate IL- 1,8 following IL-2 stimulation (Table 1). The cells in the monocyte-depleted cell preparations which are responsible for IL-2-induced IL-l/3 production migrate to the low-density fractions of discontinuous Percoll gradients (Fig. 1). This suggests that T cells and NK cells, the two major components of the low-density fractions, could be the additional IL1,8 producers. Cell sorting experiments demonstrate that IL- 1p is indeed synthesized by low-density T cells, but not NK cells, in response to IL-2 (Table 2). Although others have shown that IL- 1 is produced by murine and human T cell clones and cell lines (10, 26, 27) to our knowledge, this is the first report of IL- 1 production by freshly isolated T cells in response to any stimulus. Our results do not exclude the possibility that other non-T, non-NK NLDC may also produce IL- l/3 following IL-2 stimulation.

126

NUMEROF

ET AL.

Low-density T cells represent a minor subset (5- 10%) of peripheral blood T cells. Moreover, low-density T cells are themselves a heterogeneous mixture of T cells. We show that at least one subpopulation within this mixture, CD4+ low-density T cells, produce high levels of IL- l/3 during incubation in medium alone (Table 2). The basis for this constitutive production is unclear and may be an artifact of the cell isolation procedure, or alternatively, an indication of in vivo activation of these cells. The CD4+ low-density cells respond to IL-2 by synthesizing increased levels of IL- 1p (Table 2). These findings are consistent with the report by Acres et al. which demonstrated IL- 1 expression in a CD4+ human T cell clone (26). IL-lfi is first synthesized as a 35-kDa intracellular precursor which is cleaved at some point during or following secretion to generate the mature 17-kDa extracellular form of the molecule (2829). Both the intracellular and extracellular forms of IL-lb can be detected following the stimulation of monocytes with either LPS or IL-2 for 24 hr (3,29). Both forms are also detected in cultures of NLDC following stimulation with LPS for 24 hr (Figure 2). However, in response to IL-2, only the intracellular precursor, pro-IL- 1P, is produced. There are two possible explanations for this defect in pro-IL- l/3 processing by IL2-stimulated NLDC. One is that the pro-IL- lp which is made by NLDC in response to IL-2 differs biochemically from that which is produced by LPS-stimulated NLDC or by monocytes treated with either LPS or IL-2. However, the IL-2-inducible precursor in NLDC is precipitated by the same anti-IL-10 antibody which reacts with the pro-IL- l/3 in both LPS-stimulated NLDC (Fig. 2) and IL-2-stimulated monocytes (3). Moreover, the values we report for the molecular weight and isoelectric point of the IL-2-inducible pro-IL-l@ in NLDC are comparable to those reported for the major IL-l precursor in LPS-stimulated human monocytes (25,28-30). Thus, the pro-ILl/3 in IL-2-treated NLDC, which is neither secreted nor cleaved, is probably structurally identical to the pro-IL- 10 which is processed to the mature form of the molecule. An alternative explanation is that the IL-2-activated NLDC lack the necessary machinery for complete pro-IL- 1p processing. While the nature of this processing is not fully understood, some investigators have proposed that secreted enzymes, such as elastase and plasmin- and trypsin-like proteases, play an integral role (29, 3 1). Kostura et al. have identified a convertase activity responsible for pro-IL- 1p cleavage in the cytosols of monocytes and THP. 1 cells (32). Thus, NLDC may secrete appropriate proteases or produce the cytosol-associated convertase in response to LPS but not IL-2. CD16+ NK cells constitutively express the ~75 subunit of the IL-2 receptor (33, 34) and respond to high concentrations of IL-2 by secreting IFN-y and TNFa and developing LAK activity (4-8). They also secrete IL- 10 in response to LPS (9). Therefore, it is surprising that they neither secrete IL-lb nor even generate the intracellular precursor in response to IL-2, although they do transcribe IL-l/3 mRNA (Fig. 4). This discordance between IL- lfi transcription and translation in response to a single inducing agent is not without precedent. For example, others have shown that IL- lprimed fibroblasts and recombinant CSa-stimulated PBMC each accumulate IL- lb mRNA without translating it into protein (35,36). We were initially concerned that the interaction of anti-CD 16 antibodies with their target, the Fc-y receptor (FcRIII) on NK cells, during sorting would prime the CD 16+ cells for subsequent stimulation with IL-2. Indeed, others have found that this FcR can act as a signaling structure through which IL-2-dependent activation may be

IL-2 STIMULATES

PRO-IL-10

SYNTHESIS

IN NLDC

127

greatly enhanced (37, 38). Thus, the ability of positively selected CD 16+ cells to produce TNFa (Table 2) and transcribe IL- 1p mRNA (Fig. 4) in response to IL-2 may be due, in part, to prior triggering of the cells with the anti-FcRIII antibody. Our finding that positively selected CD 16’ cells stimulated with IL-2 do not produce the IL- 1p protein, therefore, is that much more striking. Our results reinforce the notion that the production of IL- l/3 is a complex process. Depending upon the particular stimulus and cell type, the IL- 1p gene may be transcribed but the mRNA not translated; the mRNA may be translated to form pro-IL1/I, which is neither secreted nor cleaved; or alternatively, the pro-IL- l/3 may be secreted and processed to form the mature, extracellular, 17-kDa cytokine. By analyzing and comparing these different situations, we may come closer to an understanding of which elements are essential for the individual stages of IL- l@ production. ACKNOWLEDGMENTS We gratefully acknowledge Drs. Stefan Endres, Gerhard Lonnemann, and Jos van der Meer for developing the cytokine RIA; Mark Ryan and John Daly for performing the cell sorting; and Dr. Fred Aronson, Eugene Brandon, Tim Ryan, and Dr. Teresa Barlozzari for their helpful comments and/or technical assistance.

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