Biol. Chem. Hoppe-Seyler Vol. 373, pp. 255-260, May 1992
Stimulation of Human Polymorphonuclear Leukocytes by Recombinant Human Interleukin-lß Magnus BERGENFELDT a ' b , Carina LiNDER b and Kjell OHLSSONb Departments of Surgery a and Surgical Pathophysiologyb, Lund University, Malmö General Hospital, Malmö, Sweden
(Received 28 November 1991 /16 March 1992)
Summary: Leukocyte activation is a property of systemic infection. Animal experiments indicate interleukin-1 (IL-1) as a possible modulator, while contradictory results have been reported from in-vitro stimulation of isolated leukocytes.The purpose of the present study was to investigate the activation of isolated polymorphonuclear (PMN) leukocytes in vitro by preparations of recombinant human IL-1/3 and IL-1 receptor antagonist, which in earlier studies could elicit and abrogate, respectively, a sepsis-like syndrome in rabbits. They have also been shown to influence acute phase protein synthesis in mice and rats, and release of leukocyte cathepsin G in vivo. It was found that recombinant human IL-lß elicited a dose-dependent luminol-enhanced chemiluminescence response in isolated human PMN leukocytes in the dose range 8.8xlO~ n -8.8 xlO~ 8 M. The effect could be blocked by prior treatment with the IL-1 re-
ceptor antagonist, indicating a direct effect on the specific IL-1 receptor. Preincubation by IL-1/8 enhanced the effect of a secondary challenge with phorbol 12-myristate 13-acetate or formyl-Met-Leu-Phe by 30-40% .The priming effect of rhIL-1/3 could also be blocked by the specific receptor antagonist. In this study, incubation of PMN leukocytes with rhIL-1/3 failed to induce degranulation of both azurophil (neutrophil proteinase 4/proteinase 3) and specific (lactoferrin) granules. rhIL-1/3 has been shown to induce degranulation in vivo, which is thus indicated as an indirect effect. We conclude that IL-1/3 is a direct and specific, but probably weak stimulator of the PMN leukocyte. It may also be active as a "primer" of the leukocyte.The failure to reproduce the full picture of leukocyte activation seen in vivo does, however, indicate the action of secondary mediators in sepsis.
Stimulierung menschlicher polymorphkerniger Leukozyten durch rekombinantes menschliches Interleukin-lß Zusammenfassung: Leukozytenaktivierung ist ein Merkmal systemischer Infektionen. Im Gegensatz zu kontroversen Ergebnissen an stimulierten Leukozyten in vitro weisen Tierexperimente auf Interleukin-1 (IL-1) als möglichen Modulator in vivo hin. In der hier vorliegenden Arbeit wurde daher die Aktiverung isolierter polymorphkerniger (PMN) Leukozyten in vitro durch Präparate von rekombinantem humanem IL-1/3 und IL-1-Rezeptor-Antagonist untersucht,
welche in früheren Experimenten ein sepsisähnliches Syndrom an Kaninchen hervorrufen bzw. verhindern konnten. Diese Substanzen beeinflußten auch die Synthese von Akutphasenproteinen in Mäusen und Ratten sowie die Ausschüttung von LeukozytenKathepsin G in vivo. Wir fanden, daß rekombinantes IL-lß in isolier11 ten PMN-Leukozyten im Bereich von 8.8 8.8 x 10~8M eine dosisabhängige luminolverstärkte
Abbreviations: BSA, bovine serum albumin; DMSO, dimethyl sulphoxide; ELISA, enzyme linked immunosorbent assay; fMet-Leu-Phe, formylmethionyl-leucyl-phenylalanine;HBSS, Hank's buffered salt solution; NP4,neutrophilproteinase-4/proteinase-3 (EC 3.4.21); IL-1, interleukin-1; IL-lra, interleukin-1 receptor antagonist; PBS, phosphate buffered saline; PMA, phorbol 12-myristate 13-acetate; PMN, polymorphonuclear; rhIL-1, recombinant human interleukin-1.
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256
M. Bergenfeldt, C. Linder and K. Ohlsson
Chemilumineszenz hervorruft. Durch Vorbehandlung mit IL-1-Rezeptor-Antagonist ließ sich dieser Effekt blockieren, was auf eine direkte Wirkung auf den spezifischen IL-1-Rezeptor schließen läßt. Eine Vorinkubation mit IL-1/3 verstärkte die Wirkung einer sekundären Stimulierung mit Phorbol-12myristat-13-acetat oder Formyl-Met-Leu-Phe um 30-40%. Dieser „priming"-Effekt des rekombinanten IL-1/3 konnte ebenfalls durch den spezifischen Rezeptor-Antagonisten gehemmt werden. In unserer Untersuchung war nach Inkubation von PMN-Leukozyten mit rekombinanten IL-lß jedoch keine Degranulation azurophiler (neutrale Proteinase 4/Prot-
Vol. 373 (1992)
einase 3) oder spezifischer Granula (Lactoferrin) zu beobachten. Da rekombinantes IL-lß in früheren Untersuchungen in vivo eine Degranulation bewirkte, handelt es sich hierbei wohl um einen indirekten Effekt. Wir schließen aus unseren Experimenten, daß IL-1/3 ein direkter und spezifischer, aber schwacher Stimulator für PMN-Leukozyten ist. Darüberhinaus kann es auch als Leukozyten-„Primer" wirken. Die Tatsache, daß nicht wie in vivo das vollständige Ausmaß der Leukozytenaktivierung erreicht wird, deutet jedoch auf die Wirkung weiterer Mediatoren in der Sepsis hin.
Key terms: Interleukin-1. interleukin-1 receptor antagonist, chemiluminescence, lactoferrin, leukocyte protease.
Interleukin-1 (IL-1) is the term for two potent monocyte-derived polypeptides (IL-la and IL-1/3), which mediate several systemic responses to infection like fever, anorexia, catabolism, synthesis of acute phase proteins, cortisol secretion and T-cell activation. Leukocyte activation is a property of systemic infection[1], and indirect evidence indicates IL-1 as a possible modulator. In animals, local injections of IL-1 are chemotactic on neutrophils and small intravenous doses produce fever and neutrophilia^2'3^ Larger doses of rhIL-1/3 have been shown to elicit a shocklike state with neutropenia in rabbits, which could be abrogated by administration of a specific IL-1 receptor antagonist (IL-lra)[4].The same rhIL-1/3 and rhlL-lra have been shown to influence interleukin-1-mediated release of leukocyte cathepsin G in rats, as well as acute phase protein-synthesis in rats and mice^. Direct activation of isolated PMN leukocytes has been noted in studies with purified native IL-1^~8^, while findings in later work using recombinant preparations of ILand IL-1/3 have been contradictoryl3·9-12!. The purpose of the present study was to examine the effect of rhIL-1/3 on isolated human polymorphonuclear (PMN) leukocytes with special reference to degranulation and stimulation of luminol-enhanced chemiluminescence. Preparations of rhIL-1/3 and rhlL-lra were used, that earlier have shown their potency in vivo.
Materials and Methods Chemical material Hanks buffered salt solution (HBSS) with Ca2® and Mg2® and phosphate buffered physiologic saline (PBS) were obtained from Gibco Laboratories (LifeTechnologies Inc., Grand Island, N.Y., USA). Bovine serum albumin (BSA) was the product of Sigma Chemical
Company (St. Louis, Mo, USA). DextranTSOO was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. Lymfoprep (sodium metrizoate-Ficoll) was purchased from Nyegaard (Oslo, Norway). Phorbol 12-myristate 13-acetate (PMA) from Sigma was prepared as a stock solution (3mM) in dimethylsulphoxide (DMSO) (Kebo Laboratories, Stockholm, Sweden). Luminol (5-Amino-2,3-dihydro-l,4-phthalazinedione) from Serva Feinbiochemica (Heidelberg, Germany) was prepared as a stock solution (15mM) in DMSO. Formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) from Serva was prepared as a stock solution (5mM) in HBSS with 20% DMSO. Cytochalasin B was obtained from Sigma. Specific monoclonal antibodies against neutrophil proteinase 4 (NP4/proteinase-3) were raised in our laboratory and used for ELISA of NP4 according to earlier descriptions'13·141. Lyophilized human milk lactoferrin and rabbit anti-human lactoferrin antiscrum were obtained from Sigma. Homogeneity was checked by SDS-polyacrylamide gel electrophoresis and immunoelectrophoresis, respectively. Lactoferrin was measured by electroimmunoassay according to Laurell1'5'. Dr. Robert Thompson at Synergen Inc. (Boulder, Colorado, USA) kindly provided the recombinant human IL-lß (rhIL-1/3) and IL-1 receptor antagonist (IL-lra)[16·171, which were diluted in HBSS with 0.2% BSA. The same batches of protein (containing < 0.5 units endotoxin/mg protein) were used as in earlier animal experiments'4·51. Isolation of PMN leukocytes PMN leukocytes were isolated as described by Hjorth et al.'181. Briefly, human blood was collected in heparinized tubes and mixed with an equal volume of DextranTSOO 2% in 0.9% NaCl. After sedimentation for 45 min at room temperature, 15 ml of supernatant was layered on 25 ml of Lymfoprep and centrifuged at 1350 xg for 15 min. After lysis of contaminating erythrocytes in 0.87% ammonium chloride, cells were washed twice in PBS, counted and resuspended in HBSS with 1.5% heat-inactivated serum. Purity and viability (trypan blue exclusion) were more than 98%. Recovery was typically 40-50%. Chemiluminescence experiments Luminol-enhanced chemiluminescence'191 was registered in millivolts (mV) in a LKB-Wallac luminometer model 1251 (Abo, Finland). All experiments were performed at 37 °C in a semi-darkened room. Incubation mixtures were prepared with 3 x 106 PMN leukocytes with 1.2 x 10~4M Luminol and 1.5% heat-inactivated serum in a final volume of 1.1 ml HBSS. Vials were typically incubated 15 min at 37 °C before addition of stimulant and transfer to the
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Vol. 373 (1992)
257
PMN Leukocyte Stimulation by IL-1/3
luminometer carousel. Chemiluminescence was measured at an interval of 1-4 min (integration time 0.5 s) depending on the number of samples in the run. In Exp. 1:1, rhIL-1/3 was serially diluted in HBSS with 0.2% BSA and three samples of each dilution step were prepared (Fig. 1). "Positive" and "negative" control samples were stimulated with PMAand diluent, respectively. In Exp. 1:2, groups A and B were preincubated for 5 min with IL-lra (1.8 x 10"7M) and vehicle, respectively, before addition of rhIL-l (assay concentration 0.88 x 10~9M). Groups C and D served as controls and were preincubated for 5 min with IL-lra (1.8 x 10~7M) and vehicle, respectively, before the addition of vehicle. Four replicates were run in each group (Fig. 2). In Exp. 1:3, preincubation with rhIL-1/3 before PMA-stimulation (assay concentration 10~9M) was studied. Groups A and B were preincubated with rhIL-1/3 (8.8 x 10~9M) and vehicle, respectively, for 30 min at 37°C, and PMA was then immediately added. Groups C and D received rhIL-1/3 (assay concentration 8.8 x 10~9M) and vehicle, respectively, immediately before the addition of PMA. Five replicates were run in each group (Fig. 3). Exp. 1:4 was performed to study the blocking effect of IL-lra on the effect of rhIL-1/3 preincubation and stimulation with PMA (assay concentration 10~9M). Groups A and B were preincubated for 5 min at 37°C with IL-lra (1.8xlO~ 7 M) and vehicle, respectively. After preincubation, rhIL-l (assay concentration 8.8 x 10~V) and hence PMA was added. Groups C and D served as controls and were preincubated for 5 min at 37°C with IL-lra (1.8 x 10~7M) and vehicle, respectively, before stimulation with vehicle and hence PMA. Four replicates were run in each group (Fig. 4). In Exp. 1:5, the vials (8 samples in each group) were preincubated for 15 min at 37°C with rhIL-1/3 (8.8 x 10~9M) and vehicle, respectively, before the addition of fMet-Leu-Phe (assay concentration 5 x 10~6M). Controls with fMet-Leu-Phe and vehicle alone, respectively, were run. In Exp. 1:6, the samples were preincubated for 15 min at 37°C with rhIL-1/3 (8.8 x 10~9M) and vehicle, respectively, before the addition of serially diluted fMet-Leu-Phe. Samples were run in duplicate (Fig. 5). Degranulation experiments Siliconized reaction vials with 3 x 106 polymorphonuclear leukocytes in a final volume of 1.1 m/HBSS with 1.5% heat-inactivated serum were prepared and incubated at 37 °C with rhIL-1/3 or diluent (triplicate of each). Following incubation for 0, 10, 20 or 40 min, the reactions were terminated by centrifugation at 1500 xg. The supernatants were recovered and stored at -70°C until analysis of NP4 and lactoferrin. In Exp. 2:1, the incubation was performed without Cytochalasin B. Exps. 2:2 and 2:3 contained Cytochalasin B (5 /xgV/) in the medium, and stimulation was performed with rhIL-lj3 at a concentration of 8.8 x 10~10 and 8.8 x 10~9M, respectively. Exp. 2:4 was performed with the same mixture of 3 x 106 polymorphonuclear leukocytes in a final volume of 1.1 ml HBSS with 1.5% heat-inactivated serum.Triplicate samples of rhIL-1/3 (8.8 x KT 9 M), IL-lra (1.8 x KT7M), rhIL-1/3 + IL-lra and vehicle were incubated for 0-40 min as before.
50η 40-
S
1 30-
l
ιο· Λ-
ΙΟ'13 ΙΟ' 12 ΙΟ'11 ΙΟ'10 ΙΟ'9 ΙΟ'8 ΙΟ'7 10'6 IL-1 [mol//|
Fig. 1. Dose-dependent stimulation of peak Chemiluminescence response (CL) in isolated human PMN leukocytes by IL1/3. Vertical bars denote ±SD;« = 3.
est concentration of rhIL-1/3 (8.8x10 8M) was 0.5-1% of the maximum response induced with PMA ("positive control"). Blocking effect of IL-lra. The prior addition of 1.8xlO~ 7 M IL-lra completely blocked the Chemiluminescence response induced by 8.8xlO~ 10 M rhIL-1/3, i.e. the response did not differ from the control (vehicle alone). The specific receptor antagonist alone did not show any intrinsic stimulatory or inhibitory effect compared to the vehicle alone (Fig. 2). Incubation with rhIL-l before PMA-stimulation. The addition of rhIL-1/3 before PMA had an enhancing effect on the Chemiluminescence response, i.e. vials pre-stimulated with rhIL-1/8 gave a 40% greater PMA-induced response than preincubation with vehicle alone. The response did not differ between samples preincubated for 30 min and those receiving rhIL-1/3 immediately before PMA (0 min), respectively (Fig. 3).
5040-
20 10 15
Results Dose-dependent effect ofrhIL-l . A dose-dependent Chemiluminescence response in PMN leukocytes was elicited by rhIL-1/3 in the dose range 8.8 xHT 12 8.8 x 10~8M (Fig. l).The response caused by the high-
* l
20-
30
f[min]
45
60
Fig. 2. Stimulation of Chemiluminescence in isolated human PMN leukocytes (3 x 106 cells) by 8.8 x 10~l°M IL-1/3 (O-O-O). The prior addition of a specific IL-1 receptor antagonist (1.8 x 10~7M; ·-·-·) completely blocked the response, and made it equal to the Chemiluminescence with IL-lra alone (—) or vehicle alone ( ). Vertical bars denote ± SD; n = 4.
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258
Vol. 373 (1992)
M. Bergenfeldt, C. Linderand K. Ohlsson 3000-
104
£ 2000-
I
_l
ü
10 2 -
1000-
0 30 Preincub. [min]
10'y
10-:
10'8 10'7 10'6 fMet-Leu-Phe [mol//]
Fig.3. Peak chemiluminescence response elicited in isolated human PMN leukocytes (3 x 106 cells) by addition of PMA (assay concentration 10~9M) following preincubation with IL-1/3 8.8 x 10~9M (open circles) or vehicle (bars only).
Fig. 5. Peak chemiluminescence response in isolated human PMN leukocytes (3xl0 6 cells) following preincubation with 8.8 x 10~9M IL-lj3 (closed circles) or vehicle (open circles), before subsequent stimulation with fMet-Leu-Phe.
Preincubation for 30 min and addition of rhIL-1/3 immediately before PMA gave the same effect. Vertical bars denote ±SD; « = 5.
The 30% increase in response caused by IL-lß preincubation remained the same irrespective of the fMet-Leu-Phe concentration used for subsequent stimulation (shown in logarithmic scales).
Effect of IL-lra on rhIL-lß preincubation. The enhancing effect in experiment 3 caused by addition of rhIL-1/3 before PMA could be blocked by the specific IL-1-receptor antagonist (Fig. 4). Preincubation with rhIL-lß before fMet-Leu-Phestimulation. The addition of rhIL-lß had an enhancing effect on fMet-Leu-Phe-induced chemiluminescence. Vials preincubated with rhIL-lß produced a peak chemiluminescence of 719 ± 59 mV compared to 542 ±43 mVin the control group (mean ± SEM; p
Stimulation of Human Polymorphonuclear Leukocytes by Recombinant Human Interleukin-lß Magnus BERGENFELDT a ' b , Carina LiNDER b and Kjell OHLSSONb Departments of Surgery a and Surgical Pathophysiologyb, Lund University, Malmö General Hospital, Malmö, Sweden
(Received 28 November 1991 /16 March 1992)
Summary: Leukocyte activation is a property of systemic infection. Animal experiments indicate interleukin-1 (IL-1) as a possible modulator, while contradictory results have been reported from in-vitro stimulation of isolated leukocytes.The purpose of the present study was to investigate the activation of isolated polymorphonuclear (PMN) leukocytes in vitro by preparations of recombinant human IL-1/3 and IL-1 receptor antagonist, which in earlier studies could elicit and abrogate, respectively, a sepsis-like syndrome in rabbits. They have also been shown to influence acute phase protein synthesis in mice and rats, and release of leukocyte cathepsin G in vivo. It was found that recombinant human IL-lß elicited a dose-dependent luminol-enhanced chemiluminescence response in isolated human PMN leukocytes in the dose range 8.8xlO~ n -8.8 xlO~ 8 M. The effect could be blocked by prior treatment with the IL-1 re-
ceptor antagonist, indicating a direct effect on the specific IL-1 receptor. Preincubation by IL-1/8 enhanced the effect of a secondary challenge with phorbol 12-myristate 13-acetate or formyl-Met-Leu-Phe by 30-40% .The priming effect of rhIL-1/3 could also be blocked by the specific receptor antagonist. In this study, incubation of PMN leukocytes with rhIL-1/3 failed to induce degranulation of both azurophil (neutrophil proteinase 4/proteinase 3) and specific (lactoferrin) granules. rhIL-1/3 has been shown to induce degranulation in vivo, which is thus indicated as an indirect effect. We conclude that IL-1/3 is a direct and specific, but probably weak stimulator of the PMN leukocyte. It may also be active as a "primer" of the leukocyte.The failure to reproduce the full picture of leukocyte activation seen in vivo does, however, indicate the action of secondary mediators in sepsis.
Stimulierung menschlicher polymorphkerniger Leukozyten durch rekombinantes menschliches Interleukin-lß Zusammenfassung: Leukozytenaktivierung ist ein Merkmal systemischer Infektionen. Im Gegensatz zu kontroversen Ergebnissen an stimulierten Leukozyten in vitro weisen Tierexperimente auf Interleukin-1 (IL-1) als möglichen Modulator in vivo hin. In der hier vorliegenden Arbeit wurde daher die Aktiverung isolierter polymorphkerniger (PMN) Leukozyten in vitro durch Präparate von rekombinantem humanem IL-1/3 und IL-1-Rezeptor-Antagonist untersucht,
welche in früheren Experimenten ein sepsisähnliches Syndrom an Kaninchen hervorrufen bzw. verhindern konnten. Diese Substanzen beeinflußten auch die Synthese von Akutphasenproteinen in Mäusen und Ratten sowie die Ausschüttung von LeukozytenKathepsin G in vivo. Wir fanden, daß rekombinantes IL-lß in isolier11 ten PMN-Leukozyten im Bereich von 8.8 8.8 x 10~8M eine dosisabhängige luminolverstärkte
Abbreviations: BSA, bovine serum albumin; DMSO, dimethyl sulphoxide; ELISA, enzyme linked immunosorbent assay; fMet-Leu-Phe, formylmethionyl-leucyl-phenylalanine;HBSS, Hank's buffered salt solution; NP4,neutrophilproteinase-4/proteinase-3 (EC 3.4.21); IL-1, interleukin-1; IL-lra, interleukin-1 receptor antagonist; PBS, phosphate buffered saline; PMA, phorbol 12-myristate 13-acetate; PMN, polymorphonuclear; rhIL-1, recombinant human interleukin-1.
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256
M. Bergenfeldt, C. Linder and K. Ohlsson
Chemilumineszenz hervorruft. Durch Vorbehandlung mit IL-1-Rezeptor-Antagonist ließ sich dieser Effekt blockieren, was auf eine direkte Wirkung auf den spezifischen IL-1-Rezeptor schließen läßt. Eine Vorinkubation mit IL-1/3 verstärkte die Wirkung einer sekundären Stimulierung mit Phorbol-12myristat-13-acetat oder Formyl-Met-Leu-Phe um 30-40%. Dieser „priming"-Effekt des rekombinanten IL-1/3 konnte ebenfalls durch den spezifischen Rezeptor-Antagonisten gehemmt werden. In unserer Untersuchung war nach Inkubation von PMN-Leukozyten mit rekombinanten IL-lß jedoch keine Degranulation azurophiler (neutrale Proteinase 4/Prot-
Vol. 373 (1992)
einase 3) oder spezifischer Granula (Lactoferrin) zu beobachten. Da rekombinantes IL-lß in früheren Untersuchungen in vivo eine Degranulation bewirkte, handelt es sich hierbei wohl um einen indirekten Effekt. Wir schließen aus unseren Experimenten, daß IL-1/3 ein direkter und spezifischer, aber schwacher Stimulator für PMN-Leukozyten ist. Darüberhinaus kann es auch als Leukozyten-„Primer" wirken. Die Tatsache, daß nicht wie in vivo das vollständige Ausmaß der Leukozytenaktivierung erreicht wird, deutet jedoch auf die Wirkung weiterer Mediatoren in der Sepsis hin.
Key terms: Interleukin-1. interleukin-1 receptor antagonist, chemiluminescence, lactoferrin, leukocyte protease.
Interleukin-1 (IL-1) is the term for two potent monocyte-derived polypeptides (IL-la and IL-1/3), which mediate several systemic responses to infection like fever, anorexia, catabolism, synthesis of acute phase proteins, cortisol secretion and T-cell activation. Leukocyte activation is a property of systemic infection[1], and indirect evidence indicates IL-1 as a possible modulator. In animals, local injections of IL-1 are chemotactic on neutrophils and small intravenous doses produce fever and neutrophilia^2'3^ Larger doses of rhIL-1/3 have been shown to elicit a shocklike state with neutropenia in rabbits, which could be abrogated by administration of a specific IL-1 receptor antagonist (IL-lra)[4].The same rhIL-1/3 and rhlL-lra have been shown to influence interleukin-1-mediated release of leukocyte cathepsin G in rats, as well as acute phase protein-synthesis in rats and mice^. Direct activation of isolated PMN leukocytes has been noted in studies with purified native IL-1^~8^, while findings in later work using recombinant preparations of ILand IL-1/3 have been contradictoryl3·9-12!. The purpose of the present study was to examine the effect of rhIL-1/3 on isolated human polymorphonuclear (PMN) leukocytes with special reference to degranulation and stimulation of luminol-enhanced chemiluminescence. Preparations of rhIL-1/3 and rhlL-lra were used, that earlier have shown their potency in vivo.
Materials and Methods Chemical material Hanks buffered salt solution (HBSS) with Ca2® and Mg2® and phosphate buffered physiologic saline (PBS) were obtained from Gibco Laboratories (LifeTechnologies Inc., Grand Island, N.Y., USA). Bovine serum albumin (BSA) was the product of Sigma Chemical
Company (St. Louis, Mo, USA). DextranTSOO was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. Lymfoprep (sodium metrizoate-Ficoll) was purchased from Nyegaard (Oslo, Norway). Phorbol 12-myristate 13-acetate (PMA) from Sigma was prepared as a stock solution (3mM) in dimethylsulphoxide (DMSO) (Kebo Laboratories, Stockholm, Sweden). Luminol (5-Amino-2,3-dihydro-l,4-phthalazinedione) from Serva Feinbiochemica (Heidelberg, Germany) was prepared as a stock solution (15mM) in DMSO. Formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) from Serva was prepared as a stock solution (5mM) in HBSS with 20% DMSO. Cytochalasin B was obtained from Sigma. Specific monoclonal antibodies against neutrophil proteinase 4 (NP4/proteinase-3) were raised in our laboratory and used for ELISA of NP4 according to earlier descriptions'13·141. Lyophilized human milk lactoferrin and rabbit anti-human lactoferrin antiscrum were obtained from Sigma. Homogeneity was checked by SDS-polyacrylamide gel electrophoresis and immunoelectrophoresis, respectively. Lactoferrin was measured by electroimmunoassay according to Laurell1'5'. Dr. Robert Thompson at Synergen Inc. (Boulder, Colorado, USA) kindly provided the recombinant human IL-lß (rhIL-1/3) and IL-1 receptor antagonist (IL-lra)[16·171, which were diluted in HBSS with 0.2% BSA. The same batches of protein (containing < 0.5 units endotoxin/mg protein) were used as in earlier animal experiments'4·51. Isolation of PMN leukocytes PMN leukocytes were isolated as described by Hjorth et al.'181. Briefly, human blood was collected in heparinized tubes and mixed with an equal volume of DextranTSOO 2% in 0.9% NaCl. After sedimentation for 45 min at room temperature, 15 ml of supernatant was layered on 25 ml of Lymfoprep and centrifuged at 1350 xg for 15 min. After lysis of contaminating erythrocytes in 0.87% ammonium chloride, cells were washed twice in PBS, counted and resuspended in HBSS with 1.5% heat-inactivated serum. Purity and viability (trypan blue exclusion) were more than 98%. Recovery was typically 40-50%. Chemiluminescence experiments Luminol-enhanced chemiluminescence'191 was registered in millivolts (mV) in a LKB-Wallac luminometer model 1251 (Abo, Finland). All experiments were performed at 37 °C in a semi-darkened room. Incubation mixtures were prepared with 3 x 106 PMN leukocytes with 1.2 x 10~4M Luminol and 1.5% heat-inactivated serum in a final volume of 1.1 ml HBSS. Vials were typically incubated 15 min at 37 °C before addition of stimulant and transfer to the
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257
PMN Leukocyte Stimulation by IL-1/3
luminometer carousel. Chemiluminescence was measured at an interval of 1-4 min (integration time 0.5 s) depending on the number of samples in the run. In Exp. 1:1, rhIL-1/3 was serially diluted in HBSS with 0.2% BSA and three samples of each dilution step were prepared (Fig. 1). "Positive" and "negative" control samples were stimulated with PMAand diluent, respectively. In Exp. 1:2, groups A and B were preincubated for 5 min with IL-lra (1.8 x 10"7M) and vehicle, respectively, before addition of rhIL-l (assay concentration 0.88 x 10~9M). Groups C and D served as controls and were preincubated for 5 min with IL-lra (1.8 x 10~7M) and vehicle, respectively, before the addition of vehicle. Four replicates were run in each group (Fig. 2). In Exp. 1:3, preincubation with rhIL-1/3 before PMA-stimulation (assay concentration 10~9M) was studied. Groups A and B were preincubated with rhIL-1/3 (8.8 x 10~9M) and vehicle, respectively, for 30 min at 37°C, and PMA was then immediately added. Groups C and D received rhIL-1/3 (assay concentration 8.8 x 10~9M) and vehicle, respectively, immediately before the addition of PMA. Five replicates were run in each group (Fig. 3). Exp. 1:4 was performed to study the blocking effect of IL-lra on the effect of rhIL-1/3 preincubation and stimulation with PMA (assay concentration 10~9M). Groups A and B were preincubated for 5 min at 37°C with IL-lra (1.8xlO~ 7 M) and vehicle, respectively. After preincubation, rhIL-l (assay concentration 8.8 x 10~V) and hence PMA was added. Groups C and D served as controls and were preincubated for 5 min at 37°C with IL-lra (1.8 x 10~7M) and vehicle, respectively, before stimulation with vehicle and hence PMA. Four replicates were run in each group (Fig. 4). In Exp. 1:5, the vials (8 samples in each group) were preincubated for 15 min at 37°C with rhIL-1/3 (8.8 x 10~9M) and vehicle, respectively, before the addition of fMet-Leu-Phe (assay concentration 5 x 10~6M). Controls with fMet-Leu-Phe and vehicle alone, respectively, were run. In Exp. 1:6, the samples were preincubated for 15 min at 37°C with rhIL-1/3 (8.8 x 10~9M) and vehicle, respectively, before the addition of serially diluted fMet-Leu-Phe. Samples were run in duplicate (Fig. 5). Degranulation experiments Siliconized reaction vials with 3 x 106 polymorphonuclear leukocytes in a final volume of 1.1 m/HBSS with 1.5% heat-inactivated serum were prepared and incubated at 37 °C with rhIL-1/3 or diluent (triplicate of each). Following incubation for 0, 10, 20 or 40 min, the reactions were terminated by centrifugation at 1500 xg. The supernatants were recovered and stored at -70°C until analysis of NP4 and lactoferrin. In Exp. 2:1, the incubation was performed without Cytochalasin B. Exps. 2:2 and 2:3 contained Cytochalasin B (5 /xgV/) in the medium, and stimulation was performed with rhIL-lj3 at a concentration of 8.8 x 10~10 and 8.8 x 10~9M, respectively. Exp. 2:4 was performed with the same mixture of 3 x 106 polymorphonuclear leukocytes in a final volume of 1.1 ml HBSS with 1.5% heat-inactivated serum.Triplicate samples of rhIL-1/3 (8.8 x KT 9 M), IL-lra (1.8 x KT7M), rhIL-1/3 + IL-lra and vehicle were incubated for 0-40 min as before.
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ΙΟ'13 ΙΟ' 12 ΙΟ'11 ΙΟ'10 ΙΟ'9 ΙΟ'8 ΙΟ'7 10'6 IL-1 [mol//|
Fig. 1. Dose-dependent stimulation of peak Chemiluminescence response (CL) in isolated human PMN leukocytes by IL1/3. Vertical bars denote ±SD;« = 3.
est concentration of rhIL-1/3 (8.8x10 8M) was 0.5-1% of the maximum response induced with PMA ("positive control"). Blocking effect of IL-lra. The prior addition of 1.8xlO~ 7 M IL-lra completely blocked the Chemiluminescence response induced by 8.8xlO~ 10 M rhIL-1/3, i.e. the response did not differ from the control (vehicle alone). The specific receptor antagonist alone did not show any intrinsic stimulatory or inhibitory effect compared to the vehicle alone (Fig. 2). Incubation with rhIL-l before PMA-stimulation. The addition of rhIL-1/3 before PMA had an enhancing effect on the Chemiluminescence response, i.e. vials pre-stimulated with rhIL-1/8 gave a 40% greater PMA-induced response than preincubation with vehicle alone. The response did not differ between samples preincubated for 30 min and those receiving rhIL-1/3 immediately before PMA (0 min), respectively (Fig. 3).
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Results Dose-dependent effect ofrhIL-l . A dose-dependent Chemiluminescence response in PMN leukocytes was elicited by rhIL-1/3 in the dose range 8.8 xHT 12 8.8 x 10~8M (Fig. l).The response caused by the high-
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Fig. 2. Stimulation of Chemiluminescence in isolated human PMN leukocytes (3 x 106 cells) by 8.8 x 10~l°M IL-1/3 (O-O-O). The prior addition of a specific IL-1 receptor antagonist (1.8 x 10~7M; ·-·-·) completely blocked the response, and made it equal to the Chemiluminescence with IL-lra alone (—) or vehicle alone ( ). Vertical bars denote ± SD; n = 4.
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Fig.3. Peak chemiluminescence response elicited in isolated human PMN leukocytes (3 x 106 cells) by addition of PMA (assay concentration 10~9M) following preincubation with IL-1/3 8.8 x 10~9M (open circles) or vehicle (bars only).
Fig. 5. Peak chemiluminescence response in isolated human PMN leukocytes (3xl0 6 cells) following preincubation with 8.8 x 10~9M IL-lj3 (closed circles) or vehicle (open circles), before subsequent stimulation with fMet-Leu-Phe.
Preincubation for 30 min and addition of rhIL-1/3 immediately before PMA gave the same effect. Vertical bars denote ±SD; « = 5.
The 30% increase in response caused by IL-lß preincubation remained the same irrespective of the fMet-Leu-Phe concentration used for subsequent stimulation (shown in logarithmic scales).
Effect of IL-lra on rhIL-lß preincubation. The enhancing effect in experiment 3 caused by addition of rhIL-1/3 before PMA could be blocked by the specific IL-1-receptor antagonist (Fig. 4). Preincubation with rhIL-lß before fMet-Leu-Phestimulation. The addition of rhIL-lß had an enhancing effect on fMet-Leu-Phe-induced chemiluminescence. Vials preincubated with rhIL-lß produced a peak chemiluminescence of 719 ± 59 mV compared to 542 ±43 mVin the control group (mean ± SEM; p
