Linked Activation of Nitric Oxide Synthase and Cyclooxygenase in Peripheral Monocytes of Asymptomatic Migraine Without Aura Patients

Abstract

Many reports indicate that nitric oxide (NO) could be involved in migraine without aura (MWA), an extremely diffuse clinical event. Since monocyte may be a relevant source of NO, we analysed monocyte activation in MWA patients, in a period in which they were free of symptoms. NO basal production by MWA peripheral monocytes was significantly higher than in healthy subjects (91.25 ± 8.6 μm/10⁶ cells vs. 22.6 ± 3.2 μm/10⁶ cells). Interestingly, even the release of prostaglandin E₂ (PGE₂), was higher in MWA patients than in healthy subjects (3137 ± 320 pg/10⁶ cells vs. 1531 ± 220 pg/10⁶ cells). The incubation of monocytes from healthy subjects and MWA patients with N-nitro-l-arginine methyl ester caused a marked decrease of both NO and PGE₂ release. We hypothesise that NOS and cyclooxygenase pathways in monocytes are linked and are, in MWA patients, up-regulated, even in a symptoms-free period. NO and PGE₂ hyperproduction could therefore be involved in the neurovascular modifications leading to migraine attacks.

Keywords

Migraine monocytes nitric oxide

Introduction

Nitric oxide (NO), a gas generated via the l-arginine/NO biosynthetic pathway, is released by resident and migrant inflammatory cells, such as macrophages, polymorphonuclear leucocytes (PMN) and mast cells in the microcirculation (1–6).

It has recently gained attention as new class of messenger molecule and serves a variety of functions not only in cell-mediated immunity (7) but also in the modulation of vascular-smooth muscle relaxation and vascular dilatation (8). At present there is growing interest in NO as a causative molecule of migraine pain (9). Olesen recently suggested that NO formation in the endothelium of cerebral arteries is a potential initiator of a migraine attack. Migraine attacks are associated with intra- and extracranial arterial dilatation that could be due to NO (10).

NO has therefore already made the leap forward out the shadows onto the main stage of migraine mechanics from the basic fields to the clinical ones (11–15).

Several authors also reported a cross-linking between NO and cyclooxygenase (COX) pathways (16–18). In fact, both NO and COX exist in two major isoforms: the constitutive forms (COX1, NOS1 and NOS3) that are responsible for the production of PGs, and NO involved in physiological functions; and the inducible forms (COX2 and NOS2) that are, instead, rapidly up-regulated upon appropriate stimulation (19, 20).

The enzyme COX2 is more selectively distributed in mammalian tissues than COX1, which is present in almost all tissues and cells (21). NOS1 and NOS3 produce NO for minutes per response episode, whether the stimulus is chemical, such as the neurotransmitter glutamate, or mechanical such as the pulsatile strain in arterial wall. They are constitutively expressed and transiently activated by increase of intracellular calcium/calmodulin complexes.

In contrast, NOS2 can produce NO for as long as 5 days, at least in mouse macrophages, when care is taken to replenish both the inductive stimuli and the l-arginine substrate (22). This NO synthase tightly binds calmodulin even in the absence of calcium, and is expressed by several cell types following stimulation with proinflammatory mediators.

The interaction between NOS and COX could be responsible, at least in part, for the final goal leading to the pain production in migraine.

In our previous work we were able to demonstrate a sure involvement of NO in migraine without aura (MWA) attacks (23), since we reported a marked increase of nitrite serum level in NO donor induced migraine. In addition, NO interacts with many iron-containing enzymes, such as COX2, by modifying their activity (24).

In the light of these findings we analysed in the present study the synthesis and release of NO by peripheral monocytes of MWA patients in a period in which they were without symptoms in order to evaluate the unstimulated NO release.

Since it is still unclear whether a reciprocal modulation of NO and PGE₂ synthesis and release exists or not, we also analysed in the present study the PGE₂ release by peripheral monocytes.

Patients and methods

Patients

This study was conducted in 10 patients suffering from MWA (five males, five females, mean age 40.5 ± 5.2 years), randomly recruited among the out-patients of the University ‘La Sapienza’ Headache Centre and diagnosed according to the 1988 International Headache Society (IHS) criteria (25). The mean range of migraine duration was 12.4 years (mean 3.5, max 21.6 years). There was no coexistence of tension-type headache in these MWA patients. Eight clinically healthy subjects (C), sex- and age-matched (four males, four females, mean age 33 ± 5 years) were evaluated as normal controls. The study protocol was approved by our Institutional Ethic Board and informed consent was obtained from all patients and from controls. The recommended principles of the Declaration of Helsinki, September 1989, were closely observed during this clinical research study.

Both MWA patients and controls were tested while recumbent in bed at a time when MWA patients had been asymptomatic for at least 5 days and had not taken any medication for at least 10 days.

Preparation of monocyte monolayers

Peripheral venous blood (10 ml), obtained from the two groups of subjects under study, was immediately transported to the laboratory to be processed. Heparinized blood samples were diluted with an equal volume of RPMI 1640 medium (Flow Labs, MacLean, VA). After careful mixing, the diluted blood was underlayered with 10 ml of Lymphoprep (Nyegaard, Oslo, Norway) in a 50-ml sterile conical polypropylene tube (Falcon, Oxnard, CA). The samples were centrifuged for 30 min at 400 g at room temperature, and the lymphomononuclear ring was collected and washed twice with prewarmed RPMI medium and incubated for a further 45 min in Petri dishes at 37°C in order to allow the mononuclear cells to adhere to the plastic. At the end of the incubation the supernatant containing the non-adherent cells was removed and the adherent cells were washed twice with fresh medium. The obtained monolayer consisted of 95% of monocytes as determined by phagocytosis of carbon particles and staining for non-specific esterase (26, 27). Monocytes were further incubated for 24 h at 37°C in atmosphere of 5% CO₂ with 0.4 mm sodium nitroprusside (Sigma Chemical Co., St Louis, MO) or 5 μm A23187 calcium ionophore (Sigma).

Cells incubated with culture medium alone represented the controls.

In some experiments, monocytes were incubated for 24 h in the presence of different concentrations (20–100–300 μm) of N-nitro-l-arginine methyl ester (L-NAME).

At the end of the incubation, supernatants from each experiment were collected, centrifuged at 1643 g for 15 min to remove any cell debris, and stored at −20°C in small aliquots, until the successive assay.

Cell vitality, checked by trypan blue exclusion test, was 98 ± 1.0% before incubation and 95 ± 1.2% after.

NO₂ ⁻ accumulation

NO₂ ⁻ accumulation was used as an indicator of NO production in the medium and was assayed by the Griess reaction (28). Briefly, 100 μl Griess reagent (1% sulfamide/0.1% naphthylethylene diamine dihydrochloride/2% H₃PO₄) were added to 100 μl of each supernatant in triplicate wells of a 96-well plate (Corning, NY). The plates were read using a Dynatech MR5000 ELISA plate reader (Dynatech, Chantilly, VA) at 550 nm, after 10 min of incubation at room temperature. Serial dilutions of NaNO₂ were used as standard curve. Results are expressed as μm nitrite. The interassay and intra-assay coefficients of variation of nitrite assay were < 10% and the sensitivity of nitrite determination was 1 μmol/l.

Prostaglandin E₂ radioimmunoassay

Prostaglandin E₂ (PGE₂) was measured directly in all culture media by a commercial available radioimmunoassay kit (NEN Du Pont, De Nemours, Cologno Monzese, Italy). The cross-reactivates of the anti-PGE₂ antiserum with other prostanoids are < 1% with the exception of PGE₁ = 3.7%. The limit of sensitivity for the PGE₂ is 1.3 pg/ml. The intra-assay coefficients of variation range from 2% to 7% for PGE₂ assay.

Statistical analysis

Results and calculations were done blindly by two different operators. The SAS System was used for the statistical analysis of data. Paired comparison t-test procedure was used to reveal differences between pretreatment and post-treatment values. The data were reported as mean ± sem. The mean differences, the medium and the interquartile range and the minimum and maximum values have been calculated. Values of P (for a confidence interval of 95%) < 0.005 were set as the significant level for hypothesis testing.

Results

NO synthesis and release by peripheral monocytes

Since NO is a lipid and water-soluble radical gas that reacts in water with oxygen to yield other radicals, we assessed NO synthesis and release by monocytes by measuring supernatant nitrite concentration (NO₂ ⁻). Monocytes obtained from both patients and healthy subjects spontaneously release ‘in vitro’ detectable amounts of NO. Moreover, monocytes obtained from MWA patients release in 24 h culture medium a significant higher amount of nitrite than monocytes of healthy subjects (91.25 ± 8.6 μm/10⁶ cells vs. 22.6 ± 3.2 μm/10⁶ cells).

Monocyte stimulation with 5 μm calcium ionophore and 0.4 mm sodium nitroprusside led to a significant increase of NO release from both patient and healthy subject monocytes (463 ± 52 μm/10⁶ cells vs. 440 ± 50 μm/10⁶ cells and 157 ± 22 μm/10⁶ cells vs. 153 ± 15 μm/10⁶ cells, respectively) (Table 1).

Table 1

Individual values of monocyte nitrite release in migraine without aura (MWA) patients (n = 10) and healthy controls (n = 8)

Controls			Patients
Untreated	NP	A23187	Untreated	NP	A23187
20.5	453	145	90	448.3	167
31.5	410	201	91	448	134
27.2	399.4	132	93.6	465	133
24	367.8	134	90.6	456	149.9
21.4	510	145.6	89.8	479	134
28.3	423	126.8	89	435.8	145.6
12	478	208	78	499	178
17	476	134.7	105 100.2 88.7	435 499 456.6	180 199 147

Data are expressed as μμ/10⁶ cells per 24 h. Each value represents the mean of triplicate assays.

Interestingly, no significant difference between patient and healthy monocytes was detectable after calcium ionophore and sodium nitroprusside stimulation: in fact cells of both groups under study released NO derivates at the same level (Fig. 1).

Figure 1

Nitrite accumulation in culture supernatants of monocytes obtained from healthy subjects (control group, □) and migraine patients (▪). Monocytes were incubated for 24 h at 37°C with culture medium alone (untreated) or with 0.4 mm sodium nitroprusside or 5 μm calcium ionophore A23187. Data are expressed as median of all the subjects studied ± sem. ∗Significant difference from control group (P < 0.02).

PGE₂ synthesis and release by peripheral monocytes

PGE₂ synthesis and release from monocytes of both groups under study was assayed in the supernatants of 24 h incubation. Monocytes obtained from MWA patients showed a markedly higher release than healthy subjects (3137 ± 320 pg/10⁶ cells vs. 1531 ± 220 pg/10⁶ cells) (Table 2).

Table 2

Individual values of monocyte prostaglandin E₂ (PGE₂) release in migraine without aura (MWA) patients (n = 10) and healthy controls (n = 8)

Controls			Patients
Untreated	NP	A23187	Untreated	NP	A23187
1500	3256	3768	3486	6789	5100
1520	2980	4100	2990	6324	4989
1324	3245	3245	3245	7089	4876
1378	3278	3432	3378	6200	5784
1850	3189	2987	3089	6065	4567
1567	3010	4050	3110	6156	5078
1600	2890	3765	2760	5678	4300
1530	3089	3454	3009 3200 3150	5980 6430 6043	4980 5000 4789

Data are expressed in pg/10⁶ cells per 24 h. Each value represents the mean of triplicate assays.

Stimulation with 5 μm calcium ionophore and 0.4 mm sodium nitroprusside caused a significant increase of PGE₂ synthesis and release from monocytes of both patients and healthy subjects, but patient monocytes were more active than healthy ones (Fig. 2).

Figure 2

Prostaglandin E₂ (PGE₂) synthesis and release by monocytes obtained from healthy subjects (control group, □) and migraine patients (▪). Monocytes were incubated for 24 h at 37°C with culture medium alone (untreated) or with 0.4 mm sodium nitroprusside or 5 μm calcium ionophore A23187. Data are expressed as median of all the subjects studied + sem. ∗Significant difference from control group (P < 0.001).

Effect of L-NAME on nitrite and PGE₂ release by monocytes

Incubation of monocytes of both healthy subjects and migraine patients with different concentrations (20–100–300 μm) of L-NAME dose-dependently inhibited both NO and PGE₂ release (Fig. 3 and Fig. 4).

Figure 3

Nitrite accumulation in culture medium of monocytes obtained from healthy subjects (□) and migraine without aura (MWA) patients (▪) after incubation for 24 h with different concentrations of N-nitro-l-arginine methyl ester (L-NAME). Monocytes incubated in culture medium alone represented the controls. Data are expressed as median of all the experiments + sem. ∗Significant difference from control group (P < 0.01).

Figure 4

Prostaglandin E₂ (PGE₂) synthesis and release by monocytes obtained from healthy subjects (□) and migraine without aura (MWA) (▪) patients after incubation for 24 h with different concentrations of N-nitro-l-arginine methyl ester (L-NAME). Monocytes incubated in culture medium alone represented the controls. Data are expressed as median of all the experiments + sem. ∗Significant difference from control group (P < 0.03).

On the other hand, removal of L-NAME from cell cultures and resuspending cells in fresh medium completely restored the synthesis of both the compounds. It is interesting to note that the reduction of both NO and PGE₂ release was more pronounced in monocytes of MWA patients than healthy subjects.

Discussion

Although the importance of NO synthesis and release by rodent macrophages is well established, in human monocytes/macrophages it is still controversial.

In most studies, stimulation of human macrophages with bacterial products and/or lymphokines has yielded negative results (29–32). Dugas and co-workers suggested that NO synthase could be stimulated in human monocytes by an IgE-dependent mechanism or by sequential treatments with IL-4 and interferon-gamma (IFN-γ), in some donors (33, 34). Furthermore, other studies carried out on fresh blood-stream monocytes show the release of high levels of nitrite in the culture supernatants (35). The production of NO in human monocytes/macrophages remains therefore undefined, though the antigenic NO synthases' presence is readily demonstrable.

With the present study we demonstrate not only that human monocytes spontaneously release ‘in vitro’ detectable amounts of NO, but also that in MWA patients this cell system is activated, with NO release being higher than in monocytes obtained from healthy subjects.

NO release, in both patients and healthy subjects, is further increased after stimulation with calcium ionophore or after incubation with sodium nitroprusside. This compound is a divalent anion, universally known as a NO-donor. However, it has been demonstrated that sodium nitroprusside is stable in solution, that the NO release is undetectable in absence of light and that sodium nitroprusside can be readily activated to NO by metabolism in vascular smooth muscle (36, 37).

In the light of these data we hypothesize that NO release after SNP stimulation is not only due to a spontaneous chemical degradation to NO, but also requires an active enzymatic presence.

Moreover, after SNP treatment, no difference is detectable between monocytes of the two groups under study, suggesting that NO synthesis and release cannot exceed a certain level. In fact, a larger production of NO may become self-destructive (38–40), so that is not surprising that NO production is somehow limited.

It is now clear that NO plays an important role in both central and peripheral nervous systems. NOS has been detected in all areas of the human brain, suggesting that NO plays a role in neurotransmission, as well as of either nociceptive or anti-nociceptive responses, depending on the tissue level (41). NO also seems to be involved in the regulation of the cerebral circulation (42). It is not clear how exogenous NO could be responsible for the local regulation of blood flow to the brain, as is the case of NOD migraine. However, a metabolic mechanism necessary to convert organic nitrates, such as the clinically used NOD, to biologically active forms exists mainly in the vasculature (43).

These data reinforce the hypothesis of migraine as a NO-derived disturbance. At sites of the previously hypothesized migraine ‘neurogenic inflammation’ several cell types, such as vascular smooth muscle cells, monocytes, endothelial cells and neurones, may have the capacity to generate a high concentration of inducible NO, at levels comparable to the NO released by NOD (44, 45).

It is interesting that even PGE₂ release by peripheral monocytes is higher in MWA patients than in healthy subjects. This phenomenon is particularly relevant, since it is known that NO can stimulate or inhibit prostaglandin (PG) synthesis, depending on the cell type under investigation (46). PGE₂ synthesis and release remain higher in patient monocytes even after stimulation with both calcium ionophore and sodium nitroprusside. These two compounds are also responsible for an increase of NO release from monocytes.

We can hypothesize that in human monocytes there is a positive feedback between NO and PGE₂. Moreover, the incubation of monocytes of both groups under study with L-NAME, a compound known for its inhibiting activity on nitric oxide synthases, caused a marked decrease of both nitric oxide and PGE₂ release. Therefore we suppose that the activation of COX, the enzyme leading to prostaglandin synthesis, could be mediated by nitric oxide itself.

Since activated monocytes release a higher amount of oxygen radicals (O₂ ^–, H₂O₂, OH) in the same circumstances under which they make NO (47, 48), we cannot establish whether PGE₂ release could even be mediated by peroxynitrite (OONO^–) that is formed by the combination of oxygen radicals and NO. Moreover, ONOO^– can exert even stronger pro-oxidant effects (49) and through further reaction might lead to chronic pathological conditions.

On the other hand it has been reported (50) that in a mouse monocytes/macrophages cell line RAW264.7, treated with SIN-1 (3-morpholinosydnonimine), a donor of both O₂ ^– and NO, basal and lipopolysaccharide (LPS)-stimulated production is enhanced and such enhancement appeared to be mainly, if not exclusively, due to PGE₂; in fact, an increased expression of COX2 is evident. In contrast, it has been recently demonstrated (51) that NO binding with the haeme-group of COX could result in its inactivation and in subsequent PGE₂ decrease.

In the light of our data we can hypothesize that in migraine patient monocytes, COX and NOS pathways are mutually linked and probably interact with each other, and this relationship could be important in the modulation of monocyte activation. These interactions could be responsible, at least in part, for the final goal leading to the pain production in migraine. In any case, further studies are necessary to prove this hypothesis.

Footnotes

Acknowledgements

This study was supported by grants from MURST 60% 1998.

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Linked Activation of Nitric Oxide Synthase and Cyclooxygenase in Peripheral Monocytes of Asymptomatic Migraine Without Aura Patients

Abstract

Keywords

Introduction

Patients and methods

Patients

Preparation of monocyte monolayers

NO2 − accumulation

Prostaglandin E2 radioimmunoassay

Statistical analysis

Results

NO synthesis and release by peripheral monocytes

PGE2 synthesis and release by peripheral monocytes

Effect of L-NAME on nitrite and PGE2 release by monocytes

Discussion

Footnotes

Acknowledgements

References

NO₂ ⁻ accumulation

Prostaglandin E₂ radioimmunoassay

PGE₂ synthesis and release by peripheral monocytes

Effect of L-NAME on nitrite and PGE₂ release by monocytes