Agents Actions 37 (1992)

0065-4299/92/020127-07 $1.50+0.20/0 9 1992 BirkhfiuserVerlag, Basel

Modulation of human monocyte superoxide production by recombinant interleukin-3 V. Jendrossek, S. Buth, C. Stetter and M. Gahr Universit/itskinderklinik G6ttingen, Germany


We have examined the generation of superoxide by human monocytes isolated from peripheral blood cultured in the presence of recombinant human interleukin-3 in comparison to tumor necrosis factor-~ and interferon-y. The rate of superoxide production of unstimulated and stimulated monocytes [by formylmethionyl-leucyl-phenylalanine (0.1 ~tM) and by phorbolmyristate-acetate (2 ng/ml and 200ng/ml)] decreased during the culture period in the absence of interleukin-3, whereas cells treated with interleukin-3 maintained or surpassed their initial superoxide-producing capacity. An increase of phorbolmyristateacetate- and formyl-methionyl-leucyl-phenylalanine-stimulated superoxide production of monocytes cultured with interleukin-3 compared to control cells was detected first after 24 h of monocyte culture. It was maximal after 96 h of monocyte culture. At this time the stimulated superoxide production of monocytes cultured in the presence of interleukin-3 surpassed that of interferon-7 and tumor necrosis factor-~ treated cells. Suboptimal concentrations of the stimulus phorbolmyristate-acetate (2 ng/ml) resulted in higher priming than 200 ng/ml phorbolmyristate-acetate. A dose dependence of the effect of interleukin-3 on the superoxide production was observed. Our results demonstrate that interleukin-3 primes cultured human peripheral blood monocytes for enhanced stimulated respiratory burst activity to a higher extent than interferon-7 and tumor necrosis factor-c~.


Phagocytes play an important role in host defense against invading microorganisms and possibly malignant cells both by phagocytosis and by killing of phagocytosed material using reactive oxygen species (ROS). Polymorphonuclear leukocytes (PMN) and monocytes share a multienzyme complex

Address correspondence and reprint requests to Dr. M. Gahr, Universit~itskinderklinik,Robert-Koch-Str.40, W 3400 G6ttingen, Germany

(NADPH-oxidase) with basal activity in resting cells, which can be stimulated to produce ROS by various soluble [formyl-methionyl-leucyl-phenylalanine (FMLP), phorbolmyristate acetate (PMA), fluoride] or particulate (opsonized latex beads, opsonized zymosan) stimuli. The regulation of ROS production and activation of monocytes is modulated by various cytokines acting as positive or negative regulators of the functional state of phagocytes. In inflammatory tissue, cytokines could be responsible for the fine regulation of the interaction of different cell types involved in the response to infections.

128 Interleukin-3 (IL-3), a lymphokine produced by activated T-lymphocytes, functions as a multipotent hematopoietic growth factor (multi-CSF) and has been shown to modulate mature monocytes or macrophages for increased expression of class II antigen, LFA-1 antigen, interleukin-1 mRNA and improved fungicidal activity [1 3]. The aim of our study was to determine whether IL3 is able to prime cultured human peripheral blood monocytes for enhanced respiratory burst activity after stimulation with activators of the respiratory burst oxidase to the same extent as interferon-7 (IFN-7) and tumor necrosis factor-e (TNF-c0. Materials and methods

1. Reagents and chemicals Tissue culture reagents (Dulbecco's phosphatebuffered saline (PBS), L-glutamine, Hanks balanced salt solution (HBSS), Hepes buffer, RPMI1640, penicillin and streptomycin) were from Biochrom, Berlin, FRG; all other chemicals were from Sigma, Deisenhofen, FRG. Human recombinant cytokines were obtained as follows: IFN-7 (production in Escherichia coil) was from Bioferon (Laupheim, FRG) and had a specific activity of 2.0 x 10v U/mg. TNF-c~ (production in yeast) was from Boehringer (Mannheim, FRG) with a specific activity of 2.0 x 10v U/mg. IL-3 (production in yeast) was from Genezyme (Miinchen, FRG) and had a specific activity of 3.5 x 108 U/mg. AB-serum consisted of a serum-pool from 20 healthy volunteers. Lipopolysaccharide (LPS) content of cytokines and cell culture reagents was determined by the Limulus amoebocyte lysate test (E-toxate | Sigma, Deisenhofen, FRG) as described by the manufacturers protocol [4]. Only cell culture reagents and media with LPS levels lower than 10 pg/ml were used in the experiments (1 ng LPS = 10 endotoxin units). No LPS was detected in Ficoll; PBS contained < 1 pg/ml, AB-medium < 10 pg/ml. The cytokine preparations were LPS-free (IL-3, TNF-c0 or contained < 1 pg/ml (IFN-7) in the highest concentration used in the experiments. 2. Cell isolation, cell culture and superoxide assay o f cultured monocytes Human peripheral blood monocytes from healthy volunteers were isolated from buffy coats (leuko-

Agents Actions37 (1992) cyte concentrates from human peripheral venous blood) by one-step density gradient centrifugation over Ficoll-Paque (Pharmacia fine chemicals, Uppsala, Sweden) [5]. Volunteers were 20-32-year old nonsmoking male donors without infection who had not taken medications within the previous four weeks. Mononuclear cells to be cultured were washed three times with phosphate-buffered saline (PBS) pH 7.4 without calcium and magnesium at 4~ and 140 x 9, suspended in RPMI 1640 medium supplemented with 2g/1 sodium bicarbonate, 2 mM glutamine, 100 U/ml penicillin, 100 lag/ml streptomycin and 10% (v/v) heat-inactivated pooled human AB-serum (=AB-medium) at 1.0 X 10 6 cells/ml (determined by nonspecific esterase stain (Technikon, Terrytown, USA) [6]) and plated at 1.0 x 105 monocytes/well in 96-well microtiter plates (LINBRO flat-bottomed tissue culture plates, Flow, Meckenheim, FRG). Peripheral blood mononuclear cells contained 15-58% monocytes. Monocytes were allowed to adhere for 1-2 h at 37 ~ rinsed twice with RPMI 1640, and 100 ~1 of culture medium (AB-medium) with or without cytokines was added. The remaining cells were > 90-95% monocytes and > 99% viable (as determined by trypan blue exclusion test). Monocytes were cultured in 5% CO2 at 37 ~ for four days (96 h) in the presence of AB-medium, 1-100U/ml IL-3, 100-1000U/ml IFN-7 or 100-1000 U/ml TNF-~. The superoxide (O~) production of adherent monocytes was measured directly in the microtiterplates on each day of culture as superoxide dismutase inhibitable cytochrome c reduction by a modified Pick and Mizel method [7]. The culture medium was removed, the cells were rinsed with PBS and the reaction mixture was added into the culture wells. The reaction mixture consisted of a solution of 137 laM cytochrome c type VI in HBSS supplemented with 20 mM Hepes buffer (pH 7.4) with or without 300 U/ml superoxide dismutase (SOD). Stimulation of the NADPH oxidase was performed with PMA (2 or 200 ng/ml), FMLP (0.1 gM), or buffer (unstimulated cells). The cumulative superoxide production was measured simultaneously with and 30, 60 and 120 min after the addition of the stimulus at 550 nm in a Dynatech Microtiter reader MR600 (Dynatech, Denkendorf, FRG) using absorbance at 490 nm as reference. The viability of the cells did not change during the incubation period. The superoxide production was calculated from the difference in optical densities between the wells with

Agents Actions 37 (1992) and without superoxide dismutase in nmol O2/well/120 min. Concentration response studies for IFN-7 and TNF-c~ revealed a dose-related enhancement of the stimulated and unstimulated superoxide production in IFN-y- and TNF-~treated monocytes in the range 10-1000U/1 cytokine. The dose-dependent effect of IFN-y and TNF-~ on the respiratory burst activity of monocytes has already been reported by others [8-10]. Therefore, and for conciseness, we chose for presentations only the concentration with the highest effect on superoxide production.

129 nmol 02 - / w e l l . / 120 min


3. Determination of adherent cell protein Protein was determined by the method of Bradford [11] modified by Baumgarten [12] using bovine serum albumin as a standard. The test was evaluated photometrically at 630 nm within 1 h after the addition of the dye with a Dynatech Microplate reader MR600. The calibration was performed with bovine serum albumin solutions in 120 pl PBS by adding 30 lal of the dye solution.






72 time



( hours )

Figure 1

Time dependence of IL-3 priming for unstimulated O 2production in human peripheral blood monocytes. Monocytes were cultured for 24 96 h in AB-mediumwithout (e) or with (o) 100 U/ml IL-3 and the unstimulated 02-production was determined as describedin the materials and methods section. Results are presented.asmedian out of six experimentswith blood from different donors.

4. Viability Viability of monocytes was determined by trypan blue exclusion test after 96 h of monocyte culture and no differences in viability between untreated and cytokine-treated cells were seen. After 96 h of monocyte culture, cells were morphologically still monocytes.

The effect of 100 U/ml IL-3 after 96 h culture was greater (6.1-fold increase of O z production over untreated control monocytes) than the effect of 500 U/ml I F N - 7 (4.2-fold increase) and less than that of 500 U/ml T N F - ~ (8.3-fold increase) (Tables 1 and 2).

5. Evaluation of data

2. PMA- and FMLP-stimulated O~-production of monocytes cultured with IL-3 and other cytokines

Medians and range of data were presented. Data were analyzed by the nonparametric M a n n Whitney test. Results

1. Unstimulated 02-production of monocytes cultured with IL-3 and other cytok&es Monocytes isolated from human peripheral blood cultured in AB-medium without IL-3 showed a decline of the unstimulated superoxide (O~) production during 96 h, in contrast to cells cultured in the presence of this cytokine (Fig. 1). Monocytes produced more 0 2 at all times (24, 48, 72 and 96 h) if cultured with IL-3 than cells cultured without this cytokine (Fig. 1).

The P M A - and F M L P - s t i m u l a t e d O~--production of monocytes cultured without IL-3 decreased during the culture period, whereas the opposite effect was observed with cells exposed to IL-3 (Fig. 2). The O2-production of monocytes cultured in the presence of IL-3 was greater than that of cells cultured without this cytokine. This effect was first seen after 24 h and became more obvious during the culture period (Fig. 2). With all stimuli used ( P M A 2ng/ml, P M A 200 ng/ml and F M L P 0.1 t~M) the increase of 0 2production was more pronounced with 100 U/ml IL-3 than with 500 U/ml IFN-7 and 500 U/ml T N F - ~ after 96 h (Table 1). The effects of suboptimal concentrations of the stimuli ( P M A


Agents Actions 37 (1992)

Table 1 Effect of cytokine priming on superoxide production of cultured human peripheral blood monocytes. nmol O~/well/120 min Unstimulated median

FMLP 0.1 pM






PMA 2 ng/ml median





IL-3 100 U/ml



4.48 b




IFN-7 500 U/ml

0.92 b





TNF-ct 500 U/ml



3.08 b




PMA 200 ng/ml median














Modulation of human monocyte superoxide production by recombinant interleukin-3.

We have examined the generation of superoxide by human monocytes isolated from peripheral blood cultured in the presence of recombinant human interleu...
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