Isolation and Characterization of Interleukin-1 From ... - CiteSeerX

Journal

Isolation and Characterization Bovine Polymorphonuclear Peter Brucellosis

(P.C.)

C. Canning

and

of Leukocyte

Biology

45:21-28

of Interleukin-1 Leukocytes John

and Virology of Swine (J.N.) Research Units, Research Service, United States Department

(1989)

From

D. Neill

National Animal Disease Center, of Agriculture, Ames, Iowa

Agricultural

lnterleukin-1 (IL-lot and lL-1 collectively) has been shown to be produced by a wide variety of cell types. The purpose of this study was to evaluate the ability of bovine polymorphonuciear leukocytos (PMN5) to synthesize and release IL-i-like cytokines and characterize the active molecule(s). Purified peripheral blood PMN8 were cultured for various periods of time in the presence of opsonized zymosan particles. The resulting culture supernatants exhibited IL-i activity as determined by enhanced mitogen-Induced proliferation of the D10 G4.1 murine T-helper cell line. Supernatants from nonstlmulated PMNs or PMNs stimulated for less than 6 h did not enhance D1O G4.1 proliferation. The active molecule (PMNIL-1) was isolated by using gel filtration high-performance liquid chromatography (HPLC). Further characterization of the HPLC-purified molecule by SOS-PAGE and Isoelectric focusing indicates bovine PMNIL-1 has a molecular weight of 17.8 kd and api of 4.1. Key words:

cattle,

neutrophils,

IL-i,

synthesis

INTRODUCTION Interleukin1 (IL-!) is a term used to describe a number of low molecular weight cytokines whose prois induced during inflammatory processes. The genes encoding two predominant forms of IL- 1-IL-ia (acidic p1) and IL- 113 (basic pI)-have been cloned and sequenced [19]. The primary biological activity associated with IL-! is its ability to stimulate the proliferation of T-cells in response to lectins such as concanavalin A (ConA) [21]. Interleukin1 does not directly stimulate proliferation in these cultures; rather it induces the production of interleukin-2 (IL-2), which subsequently stimulates the multiplication of the T-cells [17,21]. Numerous other biologic activities have been attributed to IL-!, including induction of fever and acute-phase proteins [5,20], induction of cytokines other than IL-2 such as interferon13 [30] and granulocyte-macrophage colony-stimulating factor [2], chemoattraction of phagocytes [3], and activation of B-cell and neutrophil functions [13,22,31]. Recombinant human IL- 1a has recently been shown by Ozaki et al. [23] to enhance the resistance of mice to bacterial challenge with Pseudomonas aeruginosa or Kiebsiella pneumoniae. In addition, administration of recombinant murine IL- 1a to mice has been shown to enhance resistance to infection with the intracellular bacterial pathogen Listeria monocytogenes [4]. A variety of cells has been shown to produce IL-!, including mononuclear phagocytes [1,27], B-cells [26], duction

© 1989 Alan

R. Liss,

Inc.

epidermal cells [16], and glial cells [8]. Recent studies by Goto et al. [9,10] and Tiku et al. [29] have demonstrated that murine, rabbit, and human polymorphonuclear leukocytes (PMNs) are also capable of synthesizing IL- 1. It is not unreasonable that cells such as PMNs which share a variety of attributes with monocytes, including phagocytic activity and a common progenitor cell, would synthesize and release IL-l-like molecules. The ability of bovine phagocytic cells to produce IL-I has not been determined previously. The purpose of the present study was to determine if bovine PMNs are capable of producing factors with IL-i activity and if so, to characterize the active molecule(s).

MATERIALS Isolation

AND METHODS

of PMN5

Eight age-matched adult Holstein-Friesian cows were used as blood donors throughout this experimentation. Blood was collected into centrifuge bottles containing acid citrate dextrose solution, and PMNs were isolated as previously described [25]. Isolated PMNs were resuspended at a density of 5 x 106 cells/mi in HEPESbuffered RPMI-1640 (Grand Island Biologics Co.,

Received Reprint P.O.

April 5. 1988; accepted requests: Peter C. Canning,

Box

70, Ames,

IA 50010.

July 26, 1988. National

Animal

Disease

Center,

22

Canning

and Neill tions

Grand Island, NY) containing 0.05% (vol/vol) gentamycm sulfate (Schering Corp., Kenilworth, NJ). Differential counts were performed by using a modified Wright’s stain [Diff-Quick, American Scientific Products, McGaw Park, IL) to assess the purity of the resulting PMN populations. Monocyte contamination was determined by nonspecific esterase staining [11]. Cell

Lines

The DlO tained from (Rockvil!e, plates (1.0 cells/mi in buffered

G4. 1 murine T-helper cell clone was obthe American Type Culture Collection MD). Cells were maintained in 24-well mlIwel!) at an initial density of 1 x l0 complete medium composed of HEPES-

RPMI

1640

medium

containing

2

mM

L-

glutamine, 10% fetal bovine serum, 50 M 2-mercaptoethanol, 0.05% (vol/vol) gentamycin sulfate, and 10% crude supernatant from ConA-stimulated rat lymphocytes containing 20 mg/ml a-methyl-D-mannopyranoside (Sigma Chemical, St. Louis, MO). Feeder spleen cells were prepared from C3HIHeJ mice. These cells were irradiated with 6,000 rads of -y-irradiation and resuspended at a density of 1 x 106 cells/ml in complete medium containing 200 pg/m! of conalbumin (Sigma Chemical, St. Louis, MO). Once every 7-10 d, the DlO 04. 1 cells were harvested and resuspended in complete medium at a density of 2 x iO cells/ml. The cells were then mixed with an equal volume of irradiated feeder cells and replated as described above. A bovine T-cell clone (BT-2) previously shown to be IL-2 dependent [24] was kindly provided by Dr. Paul Baker (Immunex Corp., Seattle, WA). These cells were maintained in maintenance medium composed of HEPES-buffered RPMI-l640 containing 2 mM L-glutamine, 1 pg/ml ConA, 10% fetal bovine serum, 50 pM 2-mercaptoethanol, 0.05% (vol/vol) gentamycin sulfate, and 10% crude supernatant from ConA-stimulated bovine lymphocytes. These cells were cultured in six-well plates (5 mI/well) at an initial density of 3.3 x l0 cells/mI. Following incubation for 48-72 h in a humidified atmosphere containing 5% CO2 in air at 37#{176}C, the cells were harvested and resuspended in maintenance medium at the initial inoculation density and replated. Preparation

of Stimulus

Opsonized zymosan was chosen as a stimulus for two reasons. First, it could be easily removed from the resulting culture supernatants, and second, it stimulates the PMNs through a “natural” phagocytic process. Zymosan A (Sigma Chemical, St. Louis, MO) was opsonized with normal bovine serum as described previously [25]. The resulting opsonized zymosan prepara-

ination

were

free of detectable

as determined

Stimulation

of PMNs

iipopolysaccharide

by limulus to Produce

lysate

contam-

assays.

IL-i

Standardized PMNs (5 X 106 cells) were added to each well of a 24-well plate. Cells were stimulated with either 0.1 ml of varying concentrations of opsonized zymosan or HEPES-buffered RPMI-1640 as a negative control. The cells were cultured in a humidified atmosphere containing 5% CO2 in air at 37#{176}C for varying periods of time. Following incubation, the PMNs were pelleted by centrifugation at 700g for 10 mm. The cell-free supernatants were collected, filtered (0.45 p.m), and stored at -20#{176}Cuntil assayed. To determine if PMNs store IL-l similar to other granule enzymes and release it upon stimulation, nonstimulated PMN cultures were prepared as described. Immediately and 18 h after initiating the culture, lysates of PMNs were obtained by sonifying PMNs (5 x 106 cells, 1 ml) for 30 sec at a setting of 1 W in a sonicator (model WlO; Heat-Systems Ultrasonic Inc., Farmingdale, NY). These cell-free lysates were filtered (0.45 iim) and stored at 20#{176}C until assayed. To determine if IL-i production by PMNs required protein synthesis, PMNs were stimulated for 18 h with opsonized zymosan in the presence and absence of the protein synthesis inhibitor puromycin (10 pg/ml). Following incubation, the culture supernatants were harvested and the puromycin removed by ultrafiltration (l0-kd exclusion, Centriprep10; Amicon, Danvers, MA). The inhibitor-free retentates were then resuspended in RPMI-1640 and assayed for IL-i activity. To control for inhibitory effects of residual puromycin on the cells used to assay for IL-i, puromycin was added to stimulated PMN culture supernatants at a final concentration of 10 pgIm!. This preparation was ultrafiltered, resuspended in RPMI-1640, and assayed for IL-i activ-

ity.

IL-i

Assay

Culture supernatants were evaluated for IL-i activity by assessing their ability to stimulate proliferation of DiO 04.1 cells [12]. Cells were suspended at a concentration of 2 x l0 cells/mi in HEPES-buffered RPMI1640 containing 2 mM glutamine, 10% fetal bovine serum, 0.05% (vol/vo!) gentamycin sulfate, and 2.5 g/ml of ConA. Aliquots (0.1 ml) of 2 x iO cells were cultured in the presence of 0. 1 ml of test samples in 96-well plates for 72 h in a humidified atmosphere containing 5% CO2 in air. Cultures were pulsed with 0.5 iCi/well of [3H]-thymidine for the final 18 h of incubation. Following incubation, the cells were deposited onto glass fiber filters and the amount of cellassociated radioactivity was determined.

Bovine

ILiI Aniy Culture

evaluating of BT-2

I4 7.2 t, ddititft f eihe I N HeI

supernatants

were

their

to directly

cells.

ability Cells

were

assayed

suspended

for IL-2

stimulate

activity

by

proliferation

at a concentration

of

8 x i04 cells/mi in HEPES-buffered RPMI medium containing 2 mM L-giutamine, 10% fetal bovine serum, 50 pM 2-mercaptoethanol, and 0.05% (vol/vol) gentamycin sulfate. Aliquots (0.1 ml) of 8 x i03 cells were cultured in 96-well plates in the presence of 0.1 ml of various dilutions of test samples or recombinant bovine IL-2 (kindly provided by Dr. Paul Baker, Immunex, Seattle, WA) as a positive control. The cells were incubated for 48 h in a humidified atmosphere containing 5% CO2 in air. Cultures were pulsed with 0.5 i.Ci/well of [3H]-thymidmne for the final 18 h of incubation. Following incubation, the cells were deposited onto glass fiber filters and the amount of cell-associated radioactivity was determined.

Purification

of the Active

From Crude

Supernatants

Molecule

(PMNIL-1)

Supernatants from zymosan-stimulated PMNs were concentrated approximately 50 x by ultrafiltration by using 10-kd exclusion filters. Following concentration, the large molecular weight molecules were removed from the mixture by ultrafiltration with 30-kd exclusion filters (Centricon-30; Amicon, Danvers, MA). Further purification of the PMNIL-l was performed by using size exclusion high-performance liquid chromatography (HPLC). Briefly, 0.2-rn! samples of concentrated supernatant were injected onto a Bio-Sil TSK-125 gel filtration column (7.5 x 300 mm; Bio-Rad Laboratories, Richmond, CA). The mobile phase consisted of phosphate-buffered saline solution (0.15 M NaC! and 50 mM phosphate, pH 7.2). The flow rate during separation was 1.0 mI/mm, and the effluent was monitored at a wavelength of 214 nm. The column was calibrated and a standard curve developed by using gel filtration standards (Bio-Rad Laboratories #151-1901, Richmond, CA). Individual peaks were collected, reconcentrated back to the injection volume by ultrafiltration, and assayed for IL- 1 activity. Characterization

PMN Produce

of PMNIL-1

Several procedures were used to physically characterize the active HPLC purified PMNIL1. To determine if the material was heat stable, an aliquot of PMNIL1 was heated to 56#{176}C for 30 mm and then tested for biological activity on the DlO G4. 1 cells. The pH stability of the purified material was determined by adding 0.1 ml of either 1 N HC1 or 1 N NaOH to alter the pH of the sample (0.1 ml) to pH 4.0 or pH 10.0, respectively. Following incubation for 15 mm, the samples were neutralized to

or

IL-i

23

1 N N5011. The

ability of these samples to stimulate proliferation of DiO G4. 1 cells was compared to that of nontreated volumematched samples of active PMNIL1. The purity and isoelectric point of active HPLCseparated PMNIL1 were determined by SDS-PAGE and isoelectric focusing (IEF) by using a PhastGel System (Pharmacia AB, Uppsala, Sweden) according to the manufacturer’s instructions. Gels containing 10-15% polyacrylamide were used for the SDS-PAGE analysis and gels with a p1 range of 3-9 were used for IEF. The gels used for SDS-PAGE analysis were calibrated and standard curves were prepared by using Dalton Mark VII SDS standards (Sigma Chemical Co., St. Louis, MO). Standard curves for IEF gels were determined by using Phast-Gel IEF 3-10 standards (Pharmacia AB, Uppsala, Sweden). Following separation, the gels were stained by using automated silver-staining procedures according to the manufacturer’s instructions.

RNA Analysis To further

investigate the ability of bovine neutrophils in response to stimulation, Northern blot analyses were performed with bovine IL-la and IL-113 RNA probes (kindly provided by Dr. Charles Maliszewski, Immunex, Seattle, WA) [18]. Briefly, total cellular RNA was isolated from PMNs stimulated 8 h with opsonized zymosan by a guanidmne HCI:CsCl centrifugation procedure described previously [14]. The resulting RNA preparation was electrophoresed on formaldehyde-denaturing agarose gels [15], blotted to nitrocellulose, and hybridized as described by Thomas [28]. The blots were probed with 32P-labeled RNA transcripts synthesized with SP6 polymerase from plasmids containing bovine IL-la and bovine IL-i13 sequences as previously described [18]. to produce

Statistical

IL-i

Analysis

The statistical significance of differences between treatment group means for either the IL-i or the IL-2 assays was determined by either an analysis of variance procedure (blocked by day) or Student’s t-test with a commercially available software package (Statview, Brainpower, Inc.) on an Apple Macintosh Plus computer.

RESULTS PMN Purity Differential counts of isolated bovine PMN populations indicated that the final cell populations were composed of 91 ±3.3% neutrophils and 8.6±3.2% eosinophils. Purified preparations of PMNs were free of contaminating mononuclear cells. The mean viability of

24

Canning

and Neill #{149} P