Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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(Journal of Leukocyte Biology. 2001;70:381-385.)
© 2001 by Society for Leukocyte Biology

Monophosphoryl lipid A stimulated up-regulation of reactive oxygen intermediates in human monocytes in vitro

D. C. Saha*, R. S. Barua*,{dagger}, M. E. Astiz{ddagger}, E. C. Rackow{ddagger} and L-J. Eales-Reynolds{dagger}

{dagger} School of Biomedical and Life Sciences, University of Surrey, Guildford, UK;
* New York Medical College, Valhalla, New York; and
{ddagger} St. Vincent’s Hospital, Harrison, New York

Correspondence: Dr. L-J. Eales-Reynolds, Senior Lecturer in Immunology, School of Biomedical and Life Sciences, University of Surrey, Guildford, GU2 7XH UK. E-mail: L.Reynolds{at}surrey.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The production of reactive oxygen and nitrogen intermediates is a common response to infectious challenge in vivo. These agents have been implicated in the modulation of cytokine responses and are produced in large amounts in response to endotoxins produced by a number of infectious agents. The antigen-presenting cell activation caused by these lipopolysacchardies (LPS) has been exploited in the use of these agents as adjuvants. In recent years, less-toxic derivatives have been sought. One such agent, monophosphoryl lipid A (MPL), has been used increasingly in vivo as an adjuvant and as a modulator of the inflammatory process. It is known that this agent modulates the inflammatory response and cytokine production. In addition, we have shown its effect on the production of reactive nitrogen intermediates. In this paper, we show that MPL stimulates the release of high levels of superoxide (O2-) and hydrogen peroxide (H2O2), the latter being greater than that seen with LPS and appearing to be related to the inability of MPL to stimulate catalase activity. When cells were pretreated with LPS or MPL and subsequently challenged with LPS, the production of O2- and H2O2 was inhibited significantly by LPS and MPL. The concentration of MPL required to induce significant hyporesponsiveness to subsequent LPS challenge was 10 times lower than that of LPS. Hyporesponsiveness was greatest when induced by 10 µg/ml MPL, the same concentration that induced the maximum release of H2O2 in primary stimulation. In addition, we have shown that following MPL pretreatment, LPS stimulation does not cause the loss of cytoplasmic I{kappa}B{alpha}, which occurs when human monocytes are cultured with LPS. From our results, we propose a model for the reduced toxicity of MPL.

Key Words: mononuclear phagocytes • lipopolysaccharide • I{kappa}B{alpha} • adjuvants


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reactive oxygen intermediates (ROI), nitric oxide (NO), and its derivatives (RNI) are toxic molecules of the immune system, which contribute to the control of microbial pathogens and tumors. Current evidence also suggests additional functions for ROI and RNI in innate and adaptive immunity, including modulation of cytokine responses and regulation of intracellular signal-transduction pathways [1 ]. In recent years, monophosphoryl lipid A (MPL), a modified form of lipopolysaccharide (LPS), has been used increasingly as an immunological adjuvant [2 3 4 ]. It has also been used to induce hyporesponsiveness to subsequent stimuli in various disease states [5 , 6 ]. It is less toxic than its parent molecule, demonstrating attenuated cytokine release and decreased, disseminated vascular coagulation and cell migration, which are usually associated with LPS [7 8 9 ]. We have shown that MPL but not LPS significantly up-regulates the expression of particulate and soluble-inducible NO synthase (NOS2) and the production of NO in human monocytes [10 ]. The reasons for this are not clear. However, monocytes generate RNI via several different pathways, which also result in the production of ROIs. LPS and MPL have been shown to increase respiratory burst activity in mice [11 12 13 ], but studies on the regulation of MPL-stimulated release of superoxide (O2-) and hydrogen peroxide (H2O2) in human monocytes have not been shown. This study examines the effect of MPL and MPL-induced hyporesponsiveness on the production of ROIs and proposes a model for the reduced, toxic effects of MPL.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
All reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless stated otherwise. The MPL and LPS from Salmonella Minnesota Re595 (phenol extracts) were dissolved in Ca2+- and Mg2+-free Dulbecco’s phosphate-buffered saline (DPBS) and diluted in phenol red-free RPMI containing 200 nM L-glutamine, 10% v/v heat-inactivated fetal calf serum (FCS), and 50 µg/ml kanamycin (Gibco-BRL, Grand Island, NY). Dilutions of both agents were sonicated at 37°C for 10 min before use.

Monocyte isolation and cell culture
Human peripheral blood mononuclear cells (MNL) were isolated from buffy coats (New York blood bank, NY) by density-gradient centrifugation as previously described [10 ]. The MNL were resuspended in RPMI [containing 10% fetal bovine serum (FBS), 200 mM L-glutamine, and 50 µg/ml kanamycin] at a concentration of 1 x 106 monocytes/ml following a differential count performed using a counter, Microdiff, Model MD16 (Coulter Corp., Miami, FL).

Adherent cells were obtained by culturing 200 µl aliquots of MNL in 96-well, flat-bottom, tissue-culture plates (Primaria, Baxter Scientific Products, Springfield, NJ) for 2 h at 37°C (5% CO2). Nonadherent cells were removed by washing in DPBS. The isolation efficiency was assessed by labeling cells recovered from replicate plates by ethylenediaminetetraacetic acid (EDTA; 0.2% w/v) treatment and cold shock (-70°C, 30 s) with anti-CD14-fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (Immunotech, Inc., Westbrook, ME). Analysis was performed using a FACScan and FACScan research software (Becton-Dickinson, Mountainview, CA). To compare the effect of MPL with LPS on the release of pro-inflammatory mediators, monocytes were incubated with different doses of these agents for 18 h (37°C, 5% CO2) as described previously [10 ]. At the end of the incubation period, the supernatants were harvested and frozen until tested for tumor necrosis factor {alpha} (TNF-{alpha}). The cells were washed once with DPBS, and O2- and H2O2 release was measured as described below. In other experiments, cells were incubated with MPL or LPS for 18 h, washed once with DPBS, and challenged with LPS (100 µg/ml, final concentration). The release of O2- and H2O2 was measured after 90 min as described below.

Measurement of TNF-{alpha}
Stimulation of TNF-{alpha} secretion by MPL and LPS was used as an internal control for stimulant activity. Supernatants from monocytes cultured with either reagents (as described above) were used to determine the presence of cytokine activity using a human TNF-{alpha} ELISA kit (Innogenetics, Antwerp, Belgium). The assays were performed according to the manufacturer’s instruction. The ELISA plates were read at 450 nm using a Bio-Tek ELISA plate reader (Model EL 311), and concentrations were determined with reference to the standard curve derived from recombinant human TNF-{alpha}. The sensitivity and limit of this assay were 8 and 320 pg/ml. All samples were tested in duplicate using at least twofold dilutions.

O2- measurement
O2- production was measured using a modification of the superoxide dismutase (SOD)-sensitive reduction of ferricytochrome C (FC), described previously [14 ]. Cells pretreated for 18 h with MPL or LPS were incubated with 180 µl FC and 20 µl DPBS or LPS (100 µg/ml, final concentration). O2- production was measured after 90 min as a change in the absorbance at 550 nm using the Bio-Tek ELISA plate reader. All assays were done in duplicate, and the production was expressed as nM O2-/2 x 105 monocytes.

H2O2 measurement
H2O2 was measured using a slight modification of the method described earlier [14 ]. Briefly, 180 µl phenol red solution [containing 816 mg NaCl, 99 mg DL(+) glucose, 20 mg phenol red, 1000 units horseradish peroxidase, and 100 ml potassium phosphate buffer] and 20 µl DPBS or LPS were added to wells containing pretreated cells as described above. The H2O2 release measured at 550 nm after 90 min using a Bio-Tek ELISA plate reader. All assays were done in duplicate, and the production was expressed as nM H2O2/2 x 105 monocytes.

Measurement of catalase activity
Catalase was measured using a slight modification of the assay described by Kashiwagi et al. [15 ]. Briefly, cells were allowed to adhere to 24-well, flat-bottom plates, washed and incubated with MPL or LPS as before. After incubation, the cells were washed once with 400 µl DPBS, and 400 µl cold, double-distilled H2O (containing 1 % v/v Triton-X 100; H2Ox) was added. The content of each well was collected, placed in an ultra-centrifuge tube, and spun at 12,000 g for 10 min at 4°C. Each supernatant (100 µL) was transferred to the wells of a 96-well, flat-bottom plate in duplicate. An equal number of control wells were set up containing 100 µl H2Ox. All the wells containing the supernatant and half the control wells were supplemented with 100 µl imidazole buffer, pH 7.4 (21.2 mM imidazole, 132 mM H2O2). The remaining wells received buffer without H2O2 to serve as a background control. The absorbance was measured using a Bio-Tek ELISA reader at 400 µm after 3 min. Results were expressed as pMoles H2O2/105 monocytes/min derived from a standard curve of catalase activity measured during the catalysis of H2O2.

Demonstration of I{kappa}B{alpha} activity
The cytoplasmic and nuclear expression of I{kappa}B{alpha} were examined using a modification of the method described by Deptala et al. [16 ]. Briefly, mononuclear leukocytes were isolated as described above, resuspended at 2.5 x 105 cells/ml in RPMI, and 200 µl was added to each well of an eight-chamber culture slide, Lab-Tek Chamber slide, (Nalgene Nunc International, Naperville, IL). After 2 h incubation (37°C, 5% CO2), the cells were washed twice, once with DPBS containing 5% FCS and once with DPBS to remove nonadherent cells. RPMI (180 µl) and 20 µl DPBS, MPL, or LPS (1 µg/ml, final concentration) were added to each chamber, and the slides incubated for 18 h (37°C, 5% CO2). The monocytes were washed once with DPBS, the wells supplemented with 200 µl RPMI, and the slides incubated for a further 45 min with or without LPS challenge (100 µg/ml, final concentration). The monocytes were washed with DPBS, and paraformaldehyde (300 µl, 1% v/v) was added to each well. After incubation on ice for 15 min, the paraformaldehyde was removed, 70% ice-cold ethanol was added, and the slides incubated for 24 h (-20°C). The slides were washed (DPBS) and incubated for 2 h at room temperature in the dark with 150 µl rabbit anti-human immunoglobulin G (IgG; 1:100 dilution, control) or rabbit polyclonal anti-human I{kappa}B{alpha} (2 µg/100 µl; Santa Cruz Biotech, Inc., Santa Cruz, CA). Slides were washed once with DPBS, and 150 µl FITC-conjugated, anti-rabbit IgG (1:50 dilution) was added to each well. After incubating for 1 h (room temperature in the dark), the chamber partition was removed, and the slides were dipped in DPBS to gently remove any antibody. The slides were flooded with propidium iodide (PI; 1 µg/ml) containing RNase (100 µg/ml) and incubated for 25 min (room temperature in the dark). After removing excess PI, the slides were fixed by flooding with DAKO fluorescent-mounting solution and examined after 5 min using a Nikon Fluorphot microscope fitted with a 100W mercury lamp.

Data analyses
Data analysis was performed using the StatView statistical program (Abacus Concepts, Inc., Berkeley, CA) using a Macintosh computer. A two-way analysis of variance (ANOVA) was used to determine the overall group differences and a one-way ANOVA was performed to determine dose differences within a group. Post hoc Fisher’s PLSD was used to determine individual differences at P < 0.05 . Figures are presented after five-point binomial data smoothing.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Stimulation of TNF-{alpha} release
TNF-{alpha} secretion was measured in supernatants of human monocytes stimulated with MPL and LPS to demonstrate their activity (Fig. 1 ). MPL stimulated the release of significantly lower levels of TNF-{alpha} than LPS in a dose-dependent manner (P<0.002). The levels of MPL-stimulated TNF-{alpha} release were not significantly different from those observed in control cultures (56±26 pg/ml), even at the highest dose examined. These results confirmed that in our hands, MPL and LPS were affecting the cells in the expected manner with respect to TNF-{alpha} release.



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  		Figure 1. Demonstration of TNF-{alpha} release by MPL- and LPS-stimulated human monocytes. Adherent peripheral blood monocytes were incubated with the indicated doses of MPL or LPS for 18 h. Supernatant TNF-{alpha} was measured using commercial ELISA kits (see Materials and Methods). Results are expressed as the mean ± SD of six experiments (P<0.002 MPL vs. LPS).

 
Production of reactive oxygen intermediates
Monocytes stimulated with MPL and LPS were assessed for their ability to secrete O2- and H2O2. Both stimulants caused the release of both intermediates from human monocytes in a dose-dependent manner (Fig. 2A and B). With respect to O2-, the effects of the two stimuli were similar. However, MPL apparently stimulated the release of significantly greater levels of H2O2 when compared with LPS (P<0.03). This apparent difference may have been a result of the greater activity of catalase, the enzyme responsible for converting H2O2 to oxygen and water, in the LPS-stimulated monocytes. Indeed, when catalase activity was measured, it was found that LPS significantly increased the activity of this enzyme in a dose-dependent manner, and MPL did not (Fig. 3 ; P<0.03). The level of activity of this enzyme in MPL-treated cells was the same as in unstimulated cells.



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  		Figure 2. Effect of MPL and LPS on the release of reactive oxygen intermediates by human monocytes. Adherent peripheral blood monocytes were incubated for 18 h with the indicated amounts of MPL or LPS, and the release of (A) O2- or (B) H2O2 was measured. Results represent the mean ± SD of seven experiments (P<0.03 MPL vs. LPS).

 


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  		Figure 3. Measurement of catalase activity in MPL- and LPS-stimulated human monocytes. Adherent peripheral blood monocytes were incubated with the indicated doses of MPL or LPS for 18 h. Catalase activity was measured using a modification of the method of Kashiwagi et al. [15] as described. The results (mean±SD of seven replicate experiments) are expressed in pmoles H2O2/105 monocytes/min (P<0.05 MPL vs. LPS).

 
Effect of pretreatment with LPS and MPL on the production of ROI upon subsequent challenge with LPS
To determine whether MPL pretreatment would induce hyporesponsiveness with respect to ROI production, cells were pretreated with a range of doses of MPL and LPS and then challenged with a high dose of LPS. MPL and LPS pretreatment of cells significantly inhibited the production of O2- by LPS-challenged monocytes. With respect to H2O2 activity, again a difference was seen between MPL and LPS. MPL pretreatment significantly inhibited H2O2 production upon challenge with doses as low as 1 µg/ml (Fig. 4 ; dose effect P<0.05). By contrast, LPS pretreatment induced significant inhibition of H2O2 production upon challenge only at the highest dose used (100 µg/ml).



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  		Figure 4. Effect of MPL and LPS pretreatment on ROI release by cells challenged with LPS. Adherent peripheral blood monocytes were incubated with the indicated amounts of MPL or LPS (n=7) for 18 h and were then challenged with 100 µg/ml LPS. The release of (A) O2- and (B) H2O2 was measured at 90 min in nM/2 x 105 monocytes. Results represent the mean ± SD of seven replicate experiments.

 
Demonstration of I{kappa}B{alpha}
To investigate this dichotomy between MPL and LPS further, we compared the effect of both the stimuli on I{kappa}B{alpha}, the known target for LPS signaling. A set of representative results is shown in Figure 5A 5B 5C . Cells cultured overnight in medium and challenged with LPS showed no evidence of cytoplasmic I{kappa}B{alpha} compared with controls, as might be expected (Fig. 5A) . In addition, cells cultured overnight with LPS and challenged with LPS showed no detectable cytoplasmic I{kappa}B{alpha} (Fig. 5B) . By contrast, cells cultured overnight with MPL and challenged with LPS showed clearly detectable levels of cytoplasmic I{kappa}B{alpha}, suggesting that pretreatment with MPL prevents I{kappa}B{alpha} breakdown upon subsequent challenge with LPS (Fig. 5C) .



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  		Figure 5. Representative examples of the effect of MPL and LPS on I{kappa}B{alpha} expression. Adherent peripheral blood monocytes were incubated with medium, MPL, or LPS (1 µg/ml, final concentration) for 18 h, washed once, and incubated for a further 45 min in RPMI alone or with LPS (100 µg/ml, final concentration) before being fixed with paraformaldehyde followed by ethanol. Cells were stained with an anti-IgG (1:100 dilution, control) or anti I{kappa}B{alpha} and second-labeled with FITC-conjugated, anti-rabbit IgG. Cells were counterstained with PI and examined using a Nikon Fluorphot microscope fitted with a 100W mercury lamp. Cytoplasmic I{kappa}B{alpha} staining is indicated by the arrows (C). (A) Monocytes cultured in medium alone for 18 h and challenged with LPS; (B) LPS-pretreated monocytes challenged with LPS; (C) MPL-pretreated monocytes challenged with LPS.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The increasing use of MPL to induce states of hyporesponsiveness in various inflammatory diseases and as an immunological adjuvant necessitates our need to understand the effect of this molecule on the immune system. It is known to be less toxic than the parent endotoxin from which it is derived. However, the nature of this reduced toxicity has not been studied extensively. In the current study, we have confirmed that MPL does not induce the release of high levels of TNF-{alpha} from human monocytes. However, it does stimulate the release of high levels of O2- and H2O2, the latter being greater than that seen with LPS. This increase in H2O2 appears to relate to the inability of MPL to stimulate catalase activity.

When cells were pretreated with MPL or LPS and subsequently challenged with LPS, the production of O2- and H2O2 was significantly inhibited. The concentration of MPL required to induce significant hyporesponsiveness to subsequent LPS challenge was 10 times lower than that of LPS. Hyporesponsiveness was greatest when induced by 10 µg/ml MPL, the same concentration that induces the maximum release of H2O2 in primary stimulation.

The induction of hyporesponsiveness to subsequent endotoxin challenge by LPS has been demonstrated previously in mice [17 ]. Only one study in humans has previously looked at the effect of hyporesponsiveness induction on ROI production in human monocytes [18 ]. LPS-treated monocytes showed a decreased ability to produce O2- and H2O2 upon subsequent challenge. By contrast, MPL failed to induce hyporesponsiveness. This apparent contradiction to the results found in our study may be explained by the fact that the authors used PMA to challenge the cells, which directly activates protein kinase C in contrast to the nuclear factor-{kappa}B (NF-{kappa}B) pathway used by MPL and LPS.

Thus, our results show that MPL induces an alteration in the redox potential of the cell through the increased production of H2O2. Several authors have suggested that regulation of nuclear transcription factors such as NF-{kappa}B (known to be activated by LPS) is dependent on the redox state of the cell and particularly on the presence of H2O2 [19 20 21 ]. However, more recent publications have suggested that NF-{kappa}B activation is not directly dependent on the redox state of the cell [22 ]. We have shown that LPS pretreatment of human monocytes (or LPS challenge of 18 h in vitro-cultured monocytes) leads to the loss of cytoplasmic I{kappa}B{alpha} (the inhibitory factor, which is degraded as a result of phosphorylation by the LPS-activated I{kappa} kinases [23 , 24 ]), and MPL pretreatment prevents this loss upon subsequent LPS challenge. Thus, in this system, the increased level of H2O2 in MPL-treated cells does not lead to NF-{kappa}B activation; in fact, it appears to stabilize the inactivated complex.

Therefore, from our results, we may propose a model for the reduced toxicity of MPL. As we have shown previously, MPL treatment of cells leads to the increased production of NO [9 ]. NO is known to have anti-inflammatory activity, and it has been proposed that it stabilizes the I{kappa}B-NF-{kappa}B complex, preventing translocation to the nucleus and transcription of genes such as TNF-{alpha} [25 ]. In addition, MPL affects catalase activity, leading to the cellular accumulation of H2O2. This may be subsequently metabolized via the glutathione-redox cycle, giving rise to high levels of oxidized glutathione, which have been shown to inhibit NF-{kappa}B activation [26 ]. Thus, upon subsequent exposure to LPS, hyporesponsiveness may occur because of a lack of NF-{kappa}B activity caused by the presence of oxidized glutathione. This prevents the transcription of genes such as those coding for TNF-{alpha}. In addition, competition for NADPH, which is common to the activity of NOS2 and NADPH oxidase, may lead to decreased cellular levels, leading to the severely decreased production of O2- and H2O2 upon subsequent challenge.

Received December 13, 2000; revised May 4, 2001; accepted May 4, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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