Exogenous nitric oxide modulates cytokine production in human leukocytes

Exogenous nitric oxide modulates cytokine production in human leukocytes

Life Sciences, Vol. 65, No. 17, pp. 1787-1794. 1999 copyright 0 1999 ElmvimS&a-a Inc. Printed in the USA. All rights rcIlQycd 0024-3205/99&~ee &at mat...

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Life Sciences, Vol. 65, No. 17, pp. 1787-1794. 1999 copyright 0 1999 ElmvimS&a-a Inc. Printed in the USA. All rights rcIlQycd 0024-3205/99&~ee &at matte


PII SO0243205(99)00431-2

EXOGENOUS NITRIC OXIDE MODULATES CYTOKINE PRODUCTION IN HUMAN LEUKOCYTES Boris-A. Kallmann, Rene Malzkom and Hubert Kolb Diabetes Research Institute, Heinrich-Heine University of Dtisseldorf, Auf m Hennekamp D-40225 DtisseldorfGermany


(Receivedin final form June 30, 1999) Summary

Exogenous nitric oxide was found to modify the pattern of cytokine secretion from human leukocytes, with similar outcome in 11 different healthy blood donors. Peripheral blood mononuclear cells (PBMC) were stimulated with phytohaemagglutinin (PHA) in the presence of increasing amounts of the NO donor S-nitroso-N-acetyl-penicillamine (SNAP). The NO donor dose-dependently enhanced IL-4 secretion into the supematant (p< 0.01). In contrast, IFNy production was not affected while IL-10 levels were slightly decreased. Comparable changes were observed when analysing cytokine mRNA levels by semiquantiative RT-PCR. The differential effect of the NO donor on II-4 versus IL-l 0 and IFN+y gene expression suggests an immunomodulatory potential of NO, which may serve to limit intlammatory responses. Key Words: nitric oxide, cytokines, human leukocytes

Nitric oxide has been shown to serve cytotoxic effector functions in immune defence (1,2) Suppression of inducible NO synthase (iNOS, NOS II) activity in immune cells compromises the resistance against several infectious pathogens (1,3), and immune defence mechanisms have been found impaired in mice with disrupted iNOS gene (4). Evidence is accumulating that NO may not only serve cytotoxic effector tinctions in immune defence. Rather, immunomodulatory functions of NO may be equally important. This view is primarily based on observation in rodents where NO from iNOS was found to suppress T cell proliferation (5-7). Furthermore, NO a%cts immune cell adhesion (8,9) and may induce or protect from apoptosis (10,ll). Of particular importance is the modulating action of NO on cytokine expression. Spleen lymphocytes of iNOS deficient mice showed enhanced production of IFNy after contact with leishmanial antigen in comparison to iNOS expressing wild type mice (4). Nitric oxide was found to decrease IFNy production in murine Thl cell clones (12). IFNy is a potent inducer of iNOS during Thl responses. It therefore has been suggested that NO from iNOS Corresponding Author: Hubert Kolb, Diabetes Research Institute, Auf m Hennekamp 65, D-40225 Dusseldorf Germany, Phone: +49-21 l-3382642, Fax: +49-21 l-3382606, Email: [email protected] de


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is part of a feedback inhibition circuit which serves to limit IFNy production and potentially deleterious cellular immune reactions (13). In the present study we tested whether these observations in mice also apply to the human immune system. When analysing Thl and Th2 type cytokine production in peripheral blood mononuclear cells (PBMC) we did not find evidence for an inhibitory effect of NO on IFNy production. Instead, we observed increased synthesis of the Th2 cytokine IL-4 after exposure to NO. Synthesis of another Th2 type cytokine, IL-10 was only weakly affected.

Methods Cell culture and reagents. Peripheral venous blood was obtained from healthy volounteers of both sexes, age range 21-45 years, and was collected in sterile 10 ml Ammonium-Heparin Monovettetubes (Sarstedt, Numbrecht, Germany). Peripheral blood mononuclear cells were isolated by FicollHypaque density gradient. Per well 200.000 cells were seeded in 96-hole tissue culture plates (Costar, Cambridge, MA) with RPMI 1640 containing 10% fetal calf serum (FCS) (Gibco, Paisley, UK). Cells were incubated (37°C 5%CO2) with 0, 0.12 or 0.4 mmovl S-nitroso-N-acetylpenicillamine (SNAP) and/or 10 &ml phytohaemagglutinin (PHA, Sigma, St.Louis, MO). SNAP was a kind gift of Dr. K. D. Kroncke (University of Dusseldorf, Dusseldorf Germany), Cell cultures were further treated with 5’-cyclic monophosphate (8-Br-cGMP) which were purchased from Sigma or 6-anilino-5,8_quinolinequinone (LY-83583) which was purchased from Calbiochem Biochemicals (San Diego, California). Supematants of cell cultures were harvested after 48 h incubation and stored frozen at -80°C until further analysis. Alternatively, cells were harvested at 24 h for RNA isolation. Cytokine measurements. Concentrations of IFNy, IL-4 and IL-IO in the supematants of cultures were determined by two-sided sandwich-ELISA (14). Briefly, 96-well Nunc-Immuno plates (MaxiSorpTM, Nunc, Denmark) were coated at 4°C with mouse anti-human IFNy monoclonal antibody (Endogen, Cambridge, MA), mouse anti-human IL-4 monoclonal antibody or rat antihuman IL-10 monoclonal antibody (PharMingen, San Diego, CA) as capture antibodies. Each incubation step was separated by repeated washing steps with PBS/O.OS% Tween20. Fifty ul of supematant samples or serial dilutions of recombinant proteins (PharMingen) as standards were added and incubated for 45 min at 37°C. Plates were then incubated with biotinylated anti-human IFNy monoclonal antibody (Endogen), IL-4 or IL-10 monoclonal antibody (PharMingen) as detection antibodies. Thereafter, 0.28-2 us/ml peroxidase-conjugate was added. For determination of residual peroxidase activity 100 ul of 1 mM ABTS containing 0.001% Hz02 was added. The optical density was measured at 405 nm against a reference wavelength of 492 nm in a Titertek Muhiskan MCC microplate reader (Flow, Meckenheim, Germany). Quantification of cytokine concentration in samples was performed using a standard curve obtained with serial dilution of recombinant cytokines mRNA analysis. RNA was isolated from peripheral blood leukocytes by the Roti-Quick-Kit (Roth, Karlsruhe, Germany). Determination and quantification of specific mRNA was performed by reverse transcriptase polymerase chain reaction (RT-PCR) as described previously for mouse and rat RNA (15,16). Reverse transcription was performed using superscript-II reverse transcriptase (200 U/ml, Gibco) and oligo (dT)-nucleotides as primer. Control experiments to check for linear relationship between amount of PCR product and amplification cycle number were performed. Specific primers were used for p-actin, LFNy, IL-4 and IL-10 (Clontech Laboratories Inc., Palo Alto, Cahf, USA). PCR products were labelled by hybridization to 32P labelled probes binding at

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sites between the primer sequences. Signals were quantified by determining phosphor stimulated luminescence (PSL) by a phosphoimager. The amount of cytokine specific PCR products were calculated as relative PSL by normalization of the measured PSL to the signal obtained for S-actin (l&16). Statistical analysis. Statistical analysis was carried out using Statview 4.01 software package on Apple Macintosh computer. All data were derived from three separate experiments of each individual. Mean values were compared by paired Student’s t-test.

Results Effect of NO on cytokineproduction. Human PBMC responded to culture with the mitogen PHA with the release of IFNy, IL-4 and IL-10 (Fig. I). Substantial interindividual differences were observed, for each of the three cytokines analysed. Culture of PBMC in the absence of PHA led to background levels of cytokines in supernatants (IFN-y < 1 &ml, IL-4 < 1 pg/ml, IL-IO < 0.1 @ml). Exposure of PBMC towards the NO donor SNAP at non toxic concentrations did not affect the production of IFNy in response to T cell stimulation. In contrast, there was significant and dose dependent upregulation of IL-4 release. At 0.12 mmoVl SNAP, IL-4 levels were increased by 30.1% (p< 0.01) and at 0.4 mmoVl SNAP the mean increase was 224.8% (p

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IL-4 (pg/ml)

IFb (ndml) 5

p<0.0001 h

IL-10 (ng/mI)






4 3-

2 L-



+ 0.12 mM + 0.4 mM SNAP SNAP


+O.l2mM SNAP

+0.4mM SNAP


+ 0.12 mM + 0.4 mM SNAP SNAP

Fig. 1

Cytokine production of PHA-stimulated PBMC in the absence or presence of SNAP. PBMC (2~10~ cells/well) of 11 healthy persons were incubated with 10 ug/ml PI-IA and 0, 0.12 or 0.4 mill SNAP for 4811. Shown are mean values of cytokine release determined in the supernatants of cell cultures (n=3). Cytokine levels in supernatants of non stimulated cultures were below the detection limit, which were 0.1 ng/ml for IFNy, 3.9 p&l for IL-4 and 0.1 @ml for IL- 10





l0.4mM SNAP

Fig. 2 Impact of SNAP on PHA-induced cytokine gene expression. PBMC were incubated with 10 &ml PHA and 0, 0.12 or 0.4 mM SNAP for 24 h. mRNA levels for IFNy, IL-4 and IL-10 were determined by RT-PCR followed by quantitation of radiolabel by phosphor-stimulated luminescence and calibrated to the amount of @ctin mRNA (set as one). Shown are mean data for five healthy individuals + SD. *, pcO.05, **, p
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IL-10 (ng/ml)

IL-4 (pglml)



Modulates Cytokine Expression


30 23 20 15

1 0.

0.1 1 10 PM 8-Br-cGMP








PM 8-Br-cGMF’





FM 8-Br-cGMP

Fig. 3 Cytokine production of PHA-stimulated PBMC after addition of 8-Br-cGMP. PBMC (2~10~ cells/well) of six healthy persons were incubated with 10 @ml PHA and 0, 0.1, 1, 10, 100 PM 8-Br-cGMP for 48 h. Shown are mean values of cy-tokine release determined in the supernatant of cell cultures (n=3).



IL-4 (&ml)




+lW LY-&%S3

+lpMLYa35%3 + O.JmMSNAP


+lW LY-S3583

+lpMLYa3S%3 + 0.4mMSNAP


+1* LY-83583

+lpMLY-S3Stu + O.JmMSNAP

Fig. 4 Cytokine production of PHA-stimulated PBMC treated with LY-83583 alone or in combination with SNAP. 1 FM LY-83583 was added to PBMC (2~10~ cells/well) of five healthy persons stimulated with 10 clg/ml PHA alone or in combination with 0.4 mM SNAP. Cell cultures were incubated for 48h. Shown are mean values of cytokine release determined in the supematant of cell cultures (n=3).

Discussion The data presented demonstrate a differential effect of the NO donor on cytokine gene expression in human PBMC. There was clear upregulation of the Th2 type cytokine IL-4 when stimulated cells


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were exposed to SNAP in addition to PHA. In contrast, no impact of the NO donor was noted on IFNy production, a key Thl type cytokine. Interestingly, NO did not exert an uniform effect on Th2 type cytokines. Although there was some increase of IL-10 secretion at the low SNAP dose there was significant suppression at the higher SNAP concentration, at which maximal enhancement of IL-4 production had been observed. IL-4 and IL-10 synergize in suppressing cellular immune reactivities, including iNOS expression (23) Hence, upregulation of IL-4 but not IL-10 synthesis will promote Th2 responses, without strongly suppressing Thl reactivity. This study did not analyse subsets of human peripheral leukocytes, but determined the overall outcome of exposure to NO in terms of the resulting cytokine balance. This approach yielded a quite uniform result, i.e. a shift towards IL-4 production in each of the 11 individual studied. In contrast, previous studies of single T cell clones remained inconclusive in that one study observed enhanced and the other suppressed IL-4 production in the presence of a NO donor (29,30). The concept that NO limits Th 1 activities by upregulating the production of antagonistic mediators is supported by our recent finding that NO induces in macrophages the gene expression of IL- 12 p40 (24). IL-l 2 p40 has been shown to form homodimers which antagonize IL-12, a major cytokine during Thl responses (25,26). Indeed, administration of IL-12(p40)2 to mice has been reported to limit but not completely suppress Thl responses (27). The molecular mechanism of the modulatory effect of NO on cytokine production is not known. In pilot experiments we were able to exclude a contribution of the classical guanylyl cyclase pathway. Numerous other targets of NO reactivity have been identified in cells, including transcription factors (3 l-34). Simple cellular model systems will be more appropriate than complex human leukocyte mixtures to address mechanistic issues. The relevance of our observations for the situation in vivo is not known. However, the experimental system used mimicked the in vivo situation in that many different immune cell types were present. Also, the concentrations of the NO donor applied are relevant in that they remain below the maximal NO concentration calculated for the vicinity of cells expressing inducible NO synthase (28). The NO-mediated upregulation of IL-4 but not of IL-IO production is expected to limit but not to My suppress Thl responses. Furthermore, NO production from iNOS also is involved in promoting wound healing (35,36). Here, upregulation of IL-4 production by NO would favour collagen production by fibroblasts (37,38). Taken together, our findings supports the view that NO from iNOS serves anti-inflammatory and repair functions in addition to the role in immune defences (13,39).


This study was supported by the Bundesminister Rir Gesundheit, by the Minister Rir Forschung und Wissenschat? des Landes Nordrhein-Westfalen, and by the Deutsche Forschungsgemeinschafi (Sondet-forschungsbereich 503). We thank J. Briiggemann for excellent technical assistance, R. Schreiner and Dr. V. Burkart for help with preparation of the manuscript.

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