Interleukin 6 primes human neutrophil and monocyte oxidative burst response

Immunology Letters, 21 (1989) 177 Elsevier IML 01225

Interleukin 6 primes human neutrophil and monocyte oxidative burst response A r s a l a n K h a r a z m i 1, H e n r i k Nielsen 1, C a t h e r i n e R e c h n i t z e r ~ a n d Klaus B e n d t z e n 2 IStatens Seruminstitut, Department of Clinical Microbiology and 2Laboratory of Medical Immunology, Department of Medicine TTA, Rigshospitalet, Copenhagen, Denmark (Received 16 February 1989; accepted 27 February 1989)

1. Summary

2. Introduction

Interleukin 6 (IL-6), a 26-kDa inducible protein, is a cytokine with multiple biological activities. This paper reports on the regulatory role of rIL-6 on the function of human polymorphonuclear and mononuclear leukocytes, a property not described previously, rIL-6 by itself did not exhibit any chemotactic activity and it could not activate these cells for an oxidative burst response. Preincubation of both cell types with rIL-6 at concentrations of 5 and 50 ng/ml primed the cells for enhanced generation of oxygen radicals following stimulation with the chemotactic peptide f-Met-Leu-Phe or the phorbol ester PMA. The enhancement of the oxidative burst response occurred both at the level of superoxide anion generation, an early step in the activation pathway, and at the level of the hydrogen peroxidemyeloperoxidase mediated response, a later step in the oxidative burst pathway. The priming ability was abolished by heat treatment of rlL-6 at 100 °C but not at 70°C. Stimulation of B cell growth and immunoglobulin production combined with enhancement of oxidative burst response of phagocytic cells by IL-6 provide an effective mechanism of fighting against invading micro-organisms.

Interleukin 6 (IL-6), also known as interferon 2 and B cell-stimulatory factor, is a 26-kDa inducible protein which is produced by many different cell types [1-4]. It has multiple biological activities and appears to play an important role in the network of interactive cytokines that are involved in the regulation of hematopoietic and immune systems [5]. IL-6 appears to interact with a variety of target cells and it is not species-specific [6]. The range of activities of IL-6 almost approaches those of interleukin 1 (ILl) [3 - 5]. Interestingly, IL-1 is a potent inducer of IL6 expression [7] and therefore it is important to distinguish the extent to which IL-1 and IL-6 biological activities overlap. Although extensive studies have been carried out on the effect of IL-6 on different cells, as reviewed by Kishimoto and Hirano [3] and by Wong and Clark [5], there are no reports on the effect of IL-6 on the function of neutrophils and monocytes, the important cells of the host defence system. In two very recent reports we have examined the effect of ILl, tumor necrosis factor (TNF), and granulocytemacrophage colony stimulating factor (GM-CSF) on the function of human phagocytes [8, 9]. This paper describes the role of IL-6 on the function of human polymorphonuclear and mononuclear leukocytes.

Key words: Interleukin 6; Neutrophil; Monocyte; Oxidativeburst Correspondence to: A. Kharazmi, Statens Seruminstitut, Department of Clinical Microbiology, Rigshospitalet 7806, Tagensvej 20, DK-2200, Copenhagen N, Denmark. 0165-2478 / 89 / $ 3.50 © 1989 Elsevier Science Publishers BN. (Biomedical Division)

177

3. Materials and Methods

3.1. 1L-6 H u m a n recombinant IL-6 was kindly provided by Dr. T. Hirano, Inst. Molecul. Cellul. Biol., Osaka University, Osaka, J a p a n [10]. The specific activity of this preparation was 1 unit/pg protein using a B9 hybridoma bioassay [11]. The endotoxin content of this preparation as measured by a sensitive Limulus amoebocyte lysate (LAL) and rocket immunoelectrophoresis assay [12] was 1.4 pg/ng rIL-6. 3.2. Neutrophils and monocytes H u m a n peripheral blood from normal donors was drawn into citrated polypropylene tubes. Polymorphonuclear and mononuclear leukocytes were separated by a two-step dextran sedimentation and sodium metrizoate Ficoll (Lymphoprep, Nyegaard, Oslo, Norway) gradient centrifugation [13]. The remaining erythrocytes were removed by hypotonic lysis. The purity of neutrophil suspension was greater than 98%. Mononuclear cell suspension contained 15-58°7o monocytes as determined by non-specific esterase staining. Cell viability as determined by trypan blue and nigrosin dye exclusion always exceeded 98%. 3.3. Chemotaxis A modified Boyden chamber assay was used as previously described for neutrophils [13] and for monocytes [14]. Briefly, the chemotaxis chambers, with a cellulose filter with 3-~m (Millipore, Bedford, MA, USA) for neutrophils or a polycarbonate filter with 5-~m pores (Nuclepore, Pleasanton, CA, USA) for monocytes, were filled with different concentrations of rIL-6 or known chemoattractants in the lower compartment. The upper c o m p a r t m e n t contained 0.5 ml of cell suspension with either 2.5 x 105 monocytes/ml or 1 x106 neutrophils/ml. The cells were preincubated with various concentratoins of rlL-6 or buffer for 30 rain at 37 *C. The assays were performed in triplicate for neutrophils and in duplicate for monocytes. After incubation for 150 min (neutrophils) or 90 min (monocytes) at 37 °C, the filters were removed from the chamber, fixed in ethanol, stained with hematoxylin and mounted on glass 178

slides. The cells that had migrated through the filter to the other side were counted by direct microscopy in 5 to 10 random fields on each filter. 3.4. Chemiluminescence A previously described chemiluminescence assay was used [16]. The assay was performed in a total volume of 5.5 ml at ambient temperature in glass scintillation vials. A Beckman l 8000 scintillation counter placed under air-conditioned thermostatcontrolled, 21 _+1 °C conditions was used in the outof-coincidence mode. A given volume of either monocyte or neutrophil suspension was preincubated with an equal volume of various concentrations of rIL-6 for 30 min at 37 °C. After preincubation, 1 ml of the cell suspension was used for the chemiluminescence assay. Each vial contained 1 ml of 5x10 s preincubated neutrophils or 2.5x10 s monocytes, phorbol myristate acetate (PMA) at 100 ng/ml, or formylated methionine-leucinephenylalanine (f-Met-Leu-Phe) at 10 -6 M, lucigenin at 1.25 #g/ml or luminol (5-amino 2,3-dihydro1,4 phthalazinedione) at 5 x 1 0 -s M. All reagents were obtained from Sigma Chemical Co., St. Louis, MO, USA. The total volume was brought up to 5.5 ml by addition of buffer. Reagents and vials were dark-adapted before use, and the experiments were performed under red light. Sequential 30-s counts were taken on each vial over a period of 60 min, and the results expressed as peak values. 3.5. f-Met-Leu-Phe binding The specific binding of f-Met-Leu-[3H]Phe to neutrophils was assessed as described by Fletcher and Gallin [17] with minor modifications. Neutrophils suspended in PBS without Ca 2++ and Mg 2+ + were preincubated with rlL-6 (50 ng/ml) or buffer for 60 min at 37 °C. Duplicate aliquots of 0.1 ml cell suspension were then incubated with fMet-Leu-[3H]Phe at 50 nM (57 Ci/mmol) in a melting ice bath to reduce internalization of the bound peptide. Nonspecific binding was measured in the presence of 1000-fold nonradioactive f-MetLeu-Phe. After 30 min incubation the cells were harvested on filters with 1 ~m retention using saline in an automatic cell harvester (Skatron, Tranby, Norway). The filters were dried and immersed in 2 ml

scintillation fluid (Opti-Fluor, Packard, Downers Grove, IL, USA). Radioactivity was measured in a Beckman L 8000 scintillation counter. Specific binding was calculated as total binding minus nonspecific binding. The nonspecific binding was less than 10% of total binding in all of the assays. 4. Results

4.1. Effect of rlL-6 on chemotaxis Experiments were first performed to determine whether rlL-6 by itself was chemotactic. When rlL-6 was used in the lower c o m p a r t m e n t of the Boyden chamber no chemotactic activity was observed over any of the cell types examined. The number of cells per field in the neutrophil assay was two for rlL-6 (50 ng/ml), three for rlL-6 (5 ng/ml), two for rlL-6 (0.5 ng/ml), and two for buffer. The number of monocytes per field was one towards 50 ng/ml of rlL-6 and one towards medium. The results on the effect of rlI~6 on neutrophil chemotaxis are shown in Table 1. Cells preincubated with rlL-6, at concentrations between 0.5 ng/ml and 50 ng/ml, exhibited a chemotactic activity towards zymosan-activated serum (ZAS) similar to the cells preincubated with buffer (170 to 178 as compared to 181 cells/field). 4.2. Effect of rlL-6 on the oxidative burst response

of neutrophils and monocytes A lucigenin-enhanced and a luminol-enhanced chemiluminescence assay were used to determine the TABLE 1 Effect of rlL-6 on neutrophil chemotaxis. rlL-6 concentrations (ng/ml)

Neutrophils/field

50 5 0.5 Buffer

172 170 178 181

The cellswere preincubated with various concentrations of rlL-6 or with buffer alone for 30 min at 37 °C. After preincubation the chemotacticactivityof the cellswas determinedagainst zymosanactivated serum.

superoxide anion production and the hydrogen peroxide-myeloperoxide (H202-MPO) mediated responses, respectively. Experiments were first performed to determine whether IL-6 could induce an oxidative burst response in neutrophils and monocytes in the absence of other stimuli. It was shown that rIL-6 by itself could not activate these two cell types to mount an oxidative burst response. The neutrophil and the monocyte responses to 50 ng/ml rIL6 were 2 x 103 cpm and 1 x 103 cpm as compared to 2 x 103 cpm induced by medium alone. In the next series of studies it was determined whether rIL-6 could influence the response of neutrophils and monocytes to known stimuli such as the chemotactic peptide f-Met-Leu-Phe or the phorbol ester PMA. The cells were preincubated with various concentrations of rIL-6 for 30 min at 37 °C. Then the cells were activated by either f-Met-Leu-Phe or PMA, and their oxygen radical generation was measured in the presence of either lucigenin or luminol. It was shown that rIL-6 at 5 ng/ml and 50 ng/ml enhanced the response of neutrophils to f-Met-LeuPhe in the lucigenin-enhanced system about twofold (Fig. 1A). In the luminol-enhanced system only a concentration of 50 ng/ml resulted in enhancement of the neutrophil response to f-Met-Leu-Phe (Fig. 1B). Kinetics of the responses both in the lucigenin- and luminol-enhanced systems showed tfiat the enhancement of the response to f-Met-LeuPhe was rapid and reached the peak within the first minute after addition of f-Met-Leu-Phe (Fig. 1A and B). The effect of rIL-6 on the response to P M A is shown in Fig. 2. rIL-6 at concentration of 50 ng/ml was able to enhance the neutrophil response to P M A by about 50%. The peak response appeared 15 to 20 min after addition of P M A to the cells. The data on the effect of rIL-6 on monocytes are presented in Fig. 3. rIL-6 at concentrations of 5 ng/ml and 50 ng/ml enhanced the response of monocytes to f-Met-Leu-Phe in the lucigeninenhanced system (Fig. 3A). The response to P M A was increased only by 50 ng/ml of IL-6 (Fig. 3B). The peak response to f-Met-Leu-Phe appeared after 5 min and to P M A after 5 - 1 0 min. Heat treatment of rIL-6 at 70°C for 15 min did not affect the priming ability of rIL-6, whereas heat treatment at 100 °C for 10 rain abolished the priming effect of rlL-6 (data not shown). 179

A

OIL-6 (50 ng/ml)

70

DIL-6 (5 ng,'ml)

A Buffer

65 60 55 50. 45. o

o × o_ o

40 35. 30. 25. 20, 15. 10, 5. 0

4

0

B

OIL-6

180

50 eg/ml

6

TIME (min)

~IL-6

8

I'0

5 ng/ml

1'4

1'2

,&Buffer

160 140 120 100 80 60 0

0

40 20

o~,

0

2

'4

6

8

1'0

1'2

1'4

TIME(min)

Fig. 1. Effect of rlL-6 on f-Met-Leu-Phe induced lucigenin-enhanced (A) or luminol-enhanced (B) neutrophil chemiluminescence. The cells were preincubated with rlL-6 at 50 ng/ml or 5 ng/ml or buffer for 30 min at 37 °C. They were then activated with f-Met-Leu-Phe at 10 -6 M. The results are from a single representative experiment performed in duplicate.

180

OIL-6

[ ] IL-6

50 ng/ml

ABuffer

5 ng/ml

500. 450. 400. 350.

o

o x

300. 250•

o 200. 150,

1O01 50

5

0 0

OIL-6

500

10

15

50 ng/ml

20 25 TIME (min)

rllL-6

30

35

5 ngtml

40

45

40

45

~Buffer

B

450 400 350

8 o x

30O 250 200 150 100

50

0# 0

5

10

15

20 25 TIME (min)

30

35

Fig. 2. Effect of rlL-6 on PMA (100 ng/ml) induced lucigenin-enhanced (A) or luminol-enhanced (B) neutrophil chemiluminescence. The experimental conditions were similar to those in Fig. l. 181

OIL-6

20-

50 ng/ml

i-IlL 6

5 ng/ml

aBuffer

A 18. 161412.

}

x

10.

o 8.

64.

2.

0 z~

OIL-6

26

50 ng/ml

;

8 TIME (rain)

rqlL-6

1'0

1'2

5 ng/ml

114

1'6

ABuffer

24 221

B

"'O

201

o

x 13.. o

181 161 141

-El .A

121

101

4. 2 0 0

2

4

8 TIME (min)

1'0

1'2

1'4

1'6

Fig. 3. Effect of rlL-6 on f-Met-Leu-Phe induced (A) or PMA induqed (B) lucigenin-enhanced monocyte chemiluminescence. The experimental conditions were similar to those in Fig. 1.

182

4.3. Effect of rlL-6 on bindin'g of f-Met-Leu-Phe to

neutrophils Cells preincubated with rlL-6 (50 ng/ml) exhibited a higher binding capacity to 3H-labelled f-MetLeu-Phe as compared to those preincubated with buffer when tested under saturation conditions. The specific binding of f-Met-Leu-[3H]Phe to the ceils preincubated with rlL-6 was 516 cpm, corresponding to 147% of the binding to control cells (350 cpm). These results are mean of 2 experiments, each performed in duplicate. 5. Discussion

This report describes the effects of rIL-6 on the chemotaxis and oxidative burst responses of human neutrophils and monocytes and on the binding of the chemotactic peptide f-Met-Leu-Phe to neutrophils. We found that rIL-6 by itself did not possess any chemotactic property for neutrophils or monocytes. Furthermore, rIL-6 alone could not activate the oxidative burst response of these cells. However, rIL-6 primed both cell types for enhanced generation of oxygen radicals when the cells were preincubated with rIL-6 and then activated by stimuli such as f-Met-Leu-Phe or PMA. Preincubation of both neutrophils and monocytes with high concentrations of rIL-6 primed these cells for enhanced generation of superoxide anion by fMet-Leu-Phe and PMA stimulated cells. The reason why high concentrations of rIL-6 were necessary to prime these cells is unknown, and to our knowledge, no one has shown that neutrophils and monocytes possess IL-6 receptors. Priming of the response to fMet-Leu-Phe was higher than that of PMA. This effect was observed in both the lucigenin-enhanced and the luminol-enhanced system. It is known that different mechanisms are involved in activation of the oxidative burst response by f-Met-Leu-Phe and PMA. f-Met-Leu-Phe binds to its receptor on the cell surface and through signal transduction activates the N A D P H oxidase, resulting in consumption of oxygen and production of moderate amounts of superoxide anion [18]. PMA stimulates protein kinase C, followed by activation of a pathway leading to generation of superoxide anion by activation of membrane N A D P H oxidase [19]. The PMA response is usually maximal, and this might be the tea-

son why in the present study rIL-6 did not prime the PMA response to the same degree as that of the fMet-Leu-Phe. This study was designed to examine the effect of rIL-6 on two different steps in the oxidative burst pathway, namely the superoxide anion production, the very first product of the oxidative burst response, and a H202-MPO mediated response, a later step in the oxidative burst pathway. A chemiluminescence assay system was used to measure the oxidative burst response of these cells. The lucigenin-enhanced system measures superoxide anion production [20, 21] and the luminol-enhanced system measures the H202-MPO mediated response [16, 22]. It was shown that rIL-6 primed both of these steps in the oxidative burst pathway. The mechanism of rIL-6 priming is not known. Bacterial endotoxins are known to prime neutrophils and monocytes for enhanced generation and release of reactive oxygen radicals [23 - 26]. It has been suggested that endotoxin priming could take place by translocation and/or partial activation of the N A D P H oxidase [24, 27]. However, the endotoxin content in the rIL-6 preparation used in this study was not at a level sufficient to prime the cells. Furthermore, heat treatment at 100 °C, which selectively destroys rIL-6 but not endotoxin, abolished the priming of the oxidative burst response. Priming of the oxidative burst response by rIL-6 could be either by alteration or modification of the cell membrane, thereby increasing contact or binding of the stimuli to the cells. Another possibility could be that rIL-6 increases the expression of receptors involved in the oxidative burst response. This alternative is supported by the demonstration of increased binding of fMet-Leu-Phe to the cells preincubated with rIL-6. It has been shown that GM-CSF, another regulatory cytokine, modulates the number and affinity of neutrophil f-Met-Leu-Phe receptors [28]. A third possibility could be partial activation of protein kinase C or N A D P H oxidase by IL-6. In all cases, we can not exclude the possibility that IL-6 increases the ability of endotoxins, which are found in all recombinant E. col#derived cytokines, to activate the N A D P H oxidase. In conclusion, these findings demonstrate that rIL-6 primes the oxidative burst response of two major cell types of the host defence system. This appears to be a new and potentially important property 183

o f r l L - 6 e v e n i f e n d o t o x i n s c o n t r i b u t e t o t h i s effect. S t i m u l a t i o n o f B cell g r o w t h a n d i m m u n o g l o b u l i n production combined with priming of phagocytic cells b y I L - 6 a n d e n d o t o x i n s p r o v i d e s a n e f f e c t i v e means of fighting invading microorganisms.

Acknowledgements We w i s h t o t h a n k A n n e A s a n o v s k i , B i r g i t t e S. N i e l s e n a n d D i a n a D a m f o r t h e i r s k i l l e d t e c h n i c a l ass i s t a n c e a n d Dr. T. H i r a n o f o r his k i n d gift o f r I L - 6 . This work was supported by the Danish Biotechnology Program, Center for Medical Immunology.

References [l] Weissenbach, J., Chernajovsky, Y, Zeevi, M., Shulman, L., Sorequ, H., Nir, U., Wallach, D., Perricaudet, M., Tiollais, P. and Revel, M. (1980) Proc. Natl. Acad. Sci. USA, 77, 7152. [2] Sehgal, P. B. and Sagar, A. D. (1980) Nature 287, 95. [3] Kishimoto, "17.and Hirano, T. (1988) Ann. Rev. Immunol. 6, 485. [4] Bendtzen, K. (1988) immunol. Letters 19, 183. [5] Wong, G. G. and Clark, S. C. (1988) lmmunol. Today 9, 137. [6] Wong, G. G., Witek, J. S., Temple, P. A., et al. (1985) Science 228, 810. [7] VanDamme, J., Opdenakker, G., Simpson, R. J., Rubira, M. R., Cayphas, S., Vink, A., Billiau, A. and VanSnick, J. (1987) J. Exp. Med. 165, 914. [8] Kharazmi, A., Nielsen, H. and Bendtzen, K. (1988) Immunobiology 177, 32. [9] Kharazmi, A., Nielsen, H. and Bendtzen, K. (1988) Imo munobiology 177, 363. [10] Hirano, T., Yasukawa, K., Harada, H., Taga, T., Watanabe, Y., Matsuda, T., Kashiwamura, S., Nakajima, K., Koyama,

184

K., lwamatsu, A., Tsumusawa, S., Sakiyama, F., Matsui, H., Takahara, Y., Taniguchi, T. and Kishimoto, T. (1986) Nature 324, 73. [11] Aarden, L. A., De Groot, E. R., Scharp, O. G. and Lansdorp, P. (1987) Eur. J. lmmunol. 17, 1411. [12] Baek, L. (1983) J. Clin. Microbiol. 17, 1013. [13] Boyum, A. (1968) Scand. J. Lab. Invest. 21, 77. [14] Kharazmi, A., Valerius, N. H. and H~iby, N. (1983) Acta Pathol. Microbiol. Immunol. Scand. Sect. C. 91, 293. [15] Nielsen, H. and Olesen Larsen, S. (1983) Acta Pathol. Microbiol. Immunol. Scand. Sect. C. 91, 109. [16] Kharazmi, A., Hoiby, N., Doting, G. and Valerius, N. H. (t984) Infect. Immun. 44, 587. [17] Fletcher, M. P. and Gallin, J. I. (1980) J. Immunol. 124, 1585. [18] McPhail, L. C. and Snyderman, R. (1983) J. Clin. Invest. 72, 192. [19] Becton, D. L, Adams, D. O. and Hamilton, T. A. (1985) J. Cell Physiol. 125, 485. [20] Weiss, S. J. and LoBuglio, A. F. (1982) Lab. Invest. 47, 5. [21] Dahlgren, C., Aniansson, H. and Magnusson, K. E. (1985) Infect. Immun. 47, 326. [22] DeChatelet, L R., Long, G. D., Shirley, P. S., Bass, D. A., Thomas, M. J., Henderson, F. W. and Cohen, M. S. (1982) J. Immunol. 129, 1589. [23] Pabst, M. J. and Johnston, R. B. (1980) J. Exp. Med. 151, 101. [24] Guthrie, L. A., McPhail, L. C., Henson, P. M. and Johnston, R. B. (1984) J. Exp. Med. 16, 1656. [25] Kharazmi, A., Rechnitzer, C., Schiotz, P. O., Jensen, T., Baek, L. and Hoiby, N. (1987) Eur. J. Clin. Invest. 17, 256. [26] Kharazmi, A., Andersen, L. W., Baek, L., Valerius, N. H., Laub, M. and Rasmussen, J. P. (1989) J. Thorac. Cardiovasc. Surg., in press. [27] Grzeskowiak, M., Bianca, V. D., Cassatella, M. A. and Rossi, E (1986) Biochem. Biophys. Res. Commun. 135, 785. [28] Weisbart, R. H., Golde, D. W. and Gasson, J. C. (1986) J. Immunol. 137, 3584.