Activation of Microglia by Histamine and Substance P Open Access

Microglia, the resident immune cells in the brain, play a pivotal role in immune surveillance of the central nervous system (CNS). Consequently, these cells are likely to play an important role in determining either the development of protective immune responses or the progression of damaging inflammation during CNS diseases [1,2,3,4]. Microglia respond to traumatic injury and the presence of infectious organisms by migrating to the site of injury, where they proliferate. When subjected to abnormal stimulation, such as neurotoxins, neuronal debris, or injury, microglia become gradually activated and produce a host of factors, including tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), interleukin-6 (IL-6), nitric oxide (NO), and reactive oxygen species (ROS). Accumulation of these proinflammatory and cytotoxic factors is directly deleterious to neurons and induces further activation of microglia, resulting in a vicious cycle [5,6]. Thus, the inhibition of microglial activation and subsequent inflammatory processes may identify novel therapeutic strategies for eliminating the deleterious effects of microglia [3]. However, the regulators and mechanisms involved in microglial activation are not fully known.

Histamine is a potent mediator of inflammation and a regulator of the innate and adaptive immune responses. Histamine induces local inflammation reactions either directly by acting on target cells or indirectly by activating other humoral and/or cellular effector systems. Histamine released from mast cells can promote Th1 and Th2 cell activation [7]. Four histamine receptors have been identified: H₁, H₂, H₃ and H₄[8,9,10,11]. Substance P (SP) is widely distributed in the central and peripheral nervous systems of vertebrates [12]. SP enhances lymphocyte proliferation, immunoglobulin production, and cytokine secretion from lymphocytes, monocytes, macrophages, and mast cells. The SP-induced release of inflammatory mediators, such as cytokines, oxygen radicals, arachidonic acid derivatives, and histamine, potentiates tissue injury and stimulates further leukocyte recruitment, thereby amplifying the inflammatory response [13]. SP can also increase vascular permeability, thus facilitating the development of an inflammatory response [14]. SP is an important proinflammatory neuropeptide that functions as an immunoneuromodulator in the brain [15,16]. This was demonstrated by inhibiting the SP-SP receptor interactions in vivo, which diminished the inflammatory responses [17], and by using mice genetically devoid of SP-SP receptor interactions [18]. Central to the ability of SP to augment inflammation is its role in modulating the function of myeloid cells, such as macrophages and dendritic cells, via SP-specific neurokinin (NK) receptors [19,20]. SP is a ligand for the NK-1 receptor and also displays some affinity for the NK-2 receptor [21]. As such, substance P has the potential to interact with a number of CNS cell types that bear substance P receptors, including neurons, astrocytes and oligodendrocytes. It has also been reported that microglia expresses the SP gene and NK-1 receptor [22]. However, little is known about the role of the neuromediators histamine and SP in brain microglia activation.

We have previously reported that the mast cell tryptase induces microglial activation and release of inflammatory factors [23]. In this study, we investigate whether the neuromediators histamine and SP can affect microglial activation and the subsequent production of proinflammatory factors.

Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum (FCS) were purchased from Gibco-BRL (Grand Island, NY, USA). Histamine, SP, and 3-(4, 5-dimethylthiazol-2-yl)-2-,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The H₁R antagonist cetirizine dihydrochloride (cetirizine), H₂R antagonist ranitidine hydrochloride (ranitidine), H₃R antagonist carcinine ditrifluoroacetate (carcinine), H₄R antagonist A943931 dihydrochloride (A943931), RP 67580 (a potent and selective tachykinin NK₁ receptor antagonist), GR 159897 (a potent and selective non-peptide neurokinin NK₂ receptor antagonist) and SB 218795 (a potent, selective and competitive non-peptide NK₃ receptor antagonist) were purchased from Tocris Bioscience (Bristol, UK). The mouse anti-rat ED8 (anti-CD11b/CD18) monoclonal antibody (a marker for complement receptor 3 of activated microglia) was purchased from AbD Serotec (Raleigh, NC, USA). Fluoroshield mounting medium with 4,6-diamidino-2-phenylindole (DAPI) was purchased from Abcam (HK, China). The Rat IL-6 Immunoassay Kit and Rat TNF-α Immunoassay Kit were obtained from R&D Systems, Inc. (Minneapolis, MN, USA). The LIVE green reactive oxygen species detection kit and MitoProbe™ JC-1 assay kit were purchased from Molecular Probes, Invitrogen (Carlsbad, CA, USA).

Rat primary microglial cells were prepared according to a previously described protocol with slight modifications [24]. Briefly, tissues from whole brains of postnatal (P1-P2) Sprague-Dawley rats were triturated, and the cells were then plated on poly-D-lysine precoated cell culture flasks in DMEM containing 10% fetal calf serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. The cultures were maintained at 37°C in a humidified atmosphere of 5% CO₂ /95% air. After reaching a confluent monolayer of glial cells (10-14 days), microglia were separated from astrocytes by shaking off for 5 h at 100 r.p.m. and replated on 24-well culture plates at a density of 10⁵ cells/cm². After plating the microglia-enriched population for 24 h, the cells were treated with lipopolysaccharide (LPS), histamine or substance P (SP) alone. The enriched microglia culture was >98% pure, as determined by OX-42-IR.

Cell viability was measured by the Thiazolyl blue (MTT) method. Briefly, cells were collected and seeded in 96-well plates at a density of 10⁵ cells/cm². After incubation for 48 h, the cells were exposed to fresh medium containing LPS (0.01 μg/ml) or various concentrations (0.001, 0.01, 0.1 and 1 µg/ml) of histamine or SP at 37°C. LPS was used as a positive control for microglial activation. After incubation for up to 24 h, 20 μL MTT tetrazolium salt (dissolved in Hank's balanced salt solution at a final concentration of 5 mg/mL) was added to each well, and the cells were placed in a CO₂ incubator for 4 h. Finally, the medium was aspirated from each well, and 150 μL of DMSO was added to dissolve the formazan crystals. The absorbance of each well was measured using a Dynatech MR5000 plate counter at test and reference wavelengths of 570 and 630 nm, respectively.

To determine the effect of histamine and SP on microglial activation, cells were fixed with 4% paraformaldehyde for 30 min. Nonspecific binding was then blocked by incubating the cells in a solution of 5% BSA and 0.1% Triton X-100 for 1 h at room temperature. The microglia were incubated with the ED8 monoclonal antibody (1:300), which recognizes complement receptor 3 (CD11b/CD18), overnight at 4°C. After three washes with PBS, the microglia were incubated with a PE-conjugated secondary antibody (1:200), and nuclei were stained with DAPI. After three washes in PBS, the cells were smeared on glass slides, and coverslips were placed and sealed with nail polish. Fluorescent images were acquired by using a confocal microscope.

The amounts of TNF-α and IL-6 in the culture medium were measured with commercial ELISA kits from R&D Systems.

The production of intracellular reactive oxygen species (ROS) was measured by DCFH oxidation. The DCFH-DA reagent passively enters cells, where it is de-acetylated by an esterase to produce the nonfluorescent DCFH. Inside the cell, DCFH reacts with ROS to form DCF, the fluorescent product. For this assay, 10 mM DCFH-DA was dissolved in methanol and diluted 500-fold in HBSS, to give a 20 μM concentration of DCFH-DA. Enriched-microglia cultures, seeded (5×10⁴) in 96-well plates, were then exposed to DCFH-DA for 1 h, followed by a 2-h treatment with HBSS containing several concentrations of LPS, histamine or substance P. After incubation, the fluorescent signal from dichlorofluorescein (DCF; excitation 495 nm, emission 529 nm) was registered every 2 min, for up to 20 min of incubation, in a Perkin-Elmer LS-5B Luminescence Spectrometer (Perkin-Elmer, Oak Brook, IL, USA) [25,26]. To calculate the amount of intracellular ROS produced, the mean signal of the control group was subtracted from that of each treatment group. Four independent sets of samples were analyzed.

The mitochondrial membrane potential (∆Ψ_m) of microglia was assessed with the MitoProbe™ JC-1 Assay Kit (Molecular Probes, Invitrogen). JC-1 exhibits potential-dependent accumulation in mitochondria, resulting in a fluorescence emission shift from green (∼529 nm) to red (∼590 nm). Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. The potential-sensitive color shift is due to the concentration-dependent formation of red fluorescent J-aggregates. Microglia, suspended in 1 ml PBS at approximately 1×10⁶ cells/ml, were incubated with 2 μM of JC-1 for 15 minutes at 37°C. The cells were washed and resuspended in 500 μl PBS and then analyzed on a flow cytometer with 488 nm excitation using emission filters appropriate for Alexa Fluor 488 dye and R-phycoerythrin. A decrease in the red/green fluorescence intensity ratio was interpreted as a loss of ∆Ψ_m, whereas an increase in the ratio was interpreted as a gain in ∆Ψ_m.

All values shown are means ± SEM. The significance of the difference between the control samples and samples treated with various drugs was determined by one-way ANOVA followed by the post-hoc least significant difference test. Differences were considered significant at P<0.05.

Cell survival, measured by 3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyl tetrazolium bromide (MTT) analysis, revealed that a 24-h incubation with LPS (0.01 μg/ml) or various concentrations (0.001, 0.01, 0.1 and 1 μg/ml) of histamine or SP had no effect on microglia viability (date not shown).

Activated microglia were detected with the monoclonal antibody ED8, which recognizes complement receptor 3 (CR3). LPS (0.01 μg/ml) was used as a positive control for microglial activation. Upon incubation with LPS (0.01 μg/ml), histamine (0.1 μg/ml) or SP (0.1 μg/ ml) for 24 hours, the ED8-positive expression (in red) was remarkably upregulated (Fig. 1). These results suggest that the neuromediators histamine and SP could activate microglia.

As microglia-mediated neuroinflammation is mainly due to an excess of proinflammatory and cytotoxic factors from activated microglia and theses factors' downstream signaling cascades, the levels of proinflammatory factors were determined. As shown in Fig. 2, incubation with LPS (0.01 μg/ml) for 24 h significantly increased the production of TNF-α (P<0.01 vs. control). Incubation with histamine at 0.001, 0.01, 0.1 and 1 μg/ml for 24 h produced a concentration-dependent increase in TNF-α levels to 572%, 965%, 1637% and 3413% of the control, respectively. Similarly, SP enhanced TNF-α production in microglia to 765%, 1374% and 2155% of the control at concentrations of 0.01, 0.1 and 1 μg/ml, respectively. Exposure to LPS (0.01 μg/ml) or histamine (0.1 μg/ml) for 30 min, 2 h, 6 h and 24 h enhanced the production of TNF-α. SP (0.1 μg/ml) exerted similar effects on TNF-α production (Fig. 3). These results indicate that histamine and SP could induce TNF-α release from microglia.

Incubation with LPS (0.01 μg/ml) for 24 h significantly enhanced the microglial production of IL-6 to 727% of the control (P<0.01). The IL-6 level was also significantly elevated, to 466% and 548% of the control, after exposure to histamine at 0.1 and 1 μg/ml, respectively. However, lower concentrations of histamine (0.001 and 0.01 μg/ml) increased the IL-6 level but not in a statistically significant way compared with the control. SP also enhanced IL-6 production, with a minimum effective dose of 0.1 μg/ml (Fig. 4). Exposure to LPS (0.01 μg/ml) and histamine (0.1 μg/ml) for 30 min, 2 h, 6 h and 24 h increased the production of IL-6, and SP (0.1 μg/ml) had similar effects on IL-6 production (Fig. 5). These results indicate that histamine and SP could induce the production of IL-6 in microglia.

The production of ROS is another important indicator of microglial activation. Therefore, the effects of different treatments on the ROS levels were examined by fluorometry. As demonstrated in Fig. 6, the incubation with LPS (0.01 μg/ml) for 2 h induced the production of ROS to 188% of the control level (P<0.01 vs. control). A 2-h incubation with histamine at 0.01, 0.1 and 1 μg/ml produced a concentration-dependent increase in the ROS level, to 115%, 131% and 148% of the control, respectively. Similarly, SP increased ROS production in microglia to 118% and 138% of the control at concentrations of 0.1 and 1 μg/ml, respectively.

We next examined the effects of antagonists of specific histamine receptors and the tachykinin receptor on inflammatory factors induced by histamine or SP and on ROS release from microglia. As shown in Fig. 7A, the H₁R antagonist Cetirizine (10 μM) and H₄R antagonist A943931 (10 µM) partially abolished the histamine (0.1 µg/ml)-induced release of TNF-α and IL-6 from microglia. In contrast, the H₂R antagonist Ranitidine (10 µM) and H₃R antagonist Carcinine ditrifluoroacetate (10 µM) had little effect on histamine-induced TNF-α and IL-6 production. However, the antagonists of H₁R, H₂R and H₄R could partially abolish histamine-induced ROS production. In this study, we also found that the selective NK-1 receptor antagonist RP67580 (10 µM), selective NK-2 receptor antagonist GR159897 (3 µM) and NK-3 receptor antagonist SB 218795 (3 µM) could partially abolished the SP (0.1 µg/ml)-induced release of TNF-α and IL-6 from microglia. The NK-1 and NK-2 receptor antagonists could also partially abolish SP-induced ROS production (Fig. 7B). These results indicate that histamine was able to induce the release of inflammatory factors from microglia primarily via H₁R and H₄R, whereas SP induced inflammatory factor release from microglia not only through the NK-1 receptor but also the NK-2 and NK-3 receptors.

As changes in the mitochondrial membrane potential (∆Ψ_m) have been shown to be involved in microglial activation and production of proinflammatory factors, the molecular probe JC-1 was used to detect the effect of histamine and SP on variations in the microglial mitochondrial membrane potential. As shown in Fig. 8, after exposure to LPS (0.01 μg/ml) for 30 min, most microglia displayed a loss or collapse of ∆Ψ_m, indicated by a fluorescence shift of JC-1 from red-orange to greenish-yellow. Similarly, incubation with histamine (0.1 μg/ml) for 30 min reduced the ∆Ψ_m of microglia. However, exposure to SP (0.1 μg/ml) for 30 min to 24 h caused no change in the ∆Ψ_m of microglia. These data indicate that histamine could induce mitochondrial membrane depolarization in microglia, whereas SP had no effect on the microglial ∆Ψ_m.

Microglial activation has been shown to be an early sign that often precedes and triggers neuronal death in chronic neurodegenerative diseases [3,5,27]. A key proinflammatory protein synthesized and released by activated microglia is TNF-α [28]. The presence of elevated levels of TNF-α has been documented in many forms of neurodegenerative disease of both chronic (e.g., AD, Parkinson's disease, and amyotrophic lateral sclerosis) and acute (e.g., stroke and head trauma) types [29]. A recent study highlighted the crucial role of IL-6, one of the proinflammatory proteins released by activated microglia, within the CNS [30]. In the CNS, IL-6 can trigger inflammation-related cellular responses, including neurogenesis, gliogenesis, cell growth, cell survival, myelination and demyelination [31,32,33]. IL-6 is normally expressed at relatively low levels in the brain, but it is elevated in the cerebral spinal fluid and brain homogenates in the presence of brain injury or inflammation [34]. Therefore, the inhibition of microglial activation and subsequent neuroinflammation may offer a therapeutic benefit for neuroinflammation-related neurodegenerative disorders. The factors responsible for the overactivation of microglia are largely undefined. Here, we demonstrate that the neuromediators histamine and SP can induce microglial activation and release of inflammatory factors.

A significant amount of brain histamine is contained not only in neurons but also in brain mast cells [35,36]. The results presented in this study demonstrate that the interaction between microglia and histamine may modulate the activation of microglia and the production of IL-6, TNF-α and ROS in the activated microglia. However, the current understanding of histamine's effect on TNF-α and IL-6 release from microglia is limited. We found that as little as 10 ng/ml of histamine was able to induce approximate increases of 5.7-fold in TNF-α release and 3.6-fold in IL-6 release from microglia, indicating that histamine is a potent stimulus of microglial activation. However, histamine did not induce the release of TNF-α in the N9 microglia cell line [37], possibly because the microglia cell line is less sensitive than primary cultured microglia. In addition, the N9 microglia cell line is mouse-derived, and there are differences in microglial activation between rat-derived and mouse-derived cells [38]. In the present study, we also found that histamine was able to induce enhanced ROS production. ROS are important molecules secreted by microglia [39]. A considerable amount of evidence suggests that oxidative stress induced by microglia-derived ROS is a major contributor to neurodegeneration [40,41]; this process involves activation of the p38 and JNK MAPKs through the upstream MKK signaling. The mitochondrial membrane potential and the downstream MAPKs have been shown to regulate microglial activation and the production of proinflammatory factors from microglia [42,43,44,45]. In the present study, we demonstrated that histamine induces mitochondrial membrane depolarization in microglia. These results suggest that histamine can induce microglial activation by reducing the mitochondrial membrane potential and regulating the downstream MAPKs.

It is now widely accepted that the neuropeptide SP can play an important role in augmenting inflammatory responses at peripheral sites. In particular, it has been demonstrated that SP can promote the immune functions of peripheral macrophages [46]. Binding of this neuropeptide to SP receptors (NK-1R) augments the production of proinflammatory monokines such as IL-1, IL-6, and TNF-α [47,48]. Furthermore, SP can induce a respiratory burst in macrophages, resulting in the production of reactive oxygen intermediates [49]. However, much less is known about the role of this neuropeptide in the initiation and/or maintenance of inflammatory responses at the site of its most ubiquitous distribution, the CNS. Luber-Narod reported that substance P enhances the secretion of tumor necrosis factor-alpha from neuroglial cells stimulated with lipopolysaccharide [50]. In the present study, we found that SP could induce the release of TNF-α and IL-6 from microglia. However, SP had no effect on the microglial mitochondrial membrane potential. Lai examined the expression of substance P and its receptor in human fetal brain microglia [51]. The ability of substance P to modulate immune cell function depends on its interaction with its receptor neurokinin-1, which is expressed in a variety of immune cells, including microglia [52]. We also found that SP could increase ROS generation in microglia; we therefore propose that SP induces the production of proinflammatory factors in microglia through elevated levels of mitochondria-derived ROS and phosphorylation by downstream MAPKs. Substance P was suggested to activate NK-1, but this has yet to be demonstrated, and a contribution of the tachykinin receptors NK-2 and NK-3 cannot be ruled out. In this study, we found that antagonists of NK-1R, NK-2R or NK-3R could partially abolish the release of SP-induced proinflammatory factors. These results suggest that not only the NK₁ receptor but the NK₂ and NK₃ receptors are also involved in SP-induced microglial activation. However, the induction of microglial ROS production by SP occurs primarily through the NK-1 and NK-2 receptors. Understanding the detailed mechanism of SP-induced microglial activation requires further study.

Our subsequent experiments showed that histamine appears to induce the release of TNF-α and IL-6 from microglia via a similar mechanism, which depends at least partially on the activation of H₁R and H₄R. It is generally thought that the stimulatory effects of histamine on the immune system are mediated by H₁R, whereas the inhibitory effects most frequently involve H₂R [53]. This concept has also been supported by a model of multiple sclerosis (MS), which showed that the dual effects of histamine are due to different pathophysiological features of four histamine receptors. Thus, H₁R and H₄R may participate in exacerbating the disease, and H₂R and H₃R may be effective in ameliorating the disease [54]. These results indicate that histamine most likely exerts a proinflammatory effect on microglia via H₁R and H₄R. However, we found that histamine induces ROS production mainly through H₁R, H₂R and H₄R; thus, this might involve a different mechanism from that of proinflammatory factor release.

In summary, we have shown here that the neuromediators histamine and SP can induce microglial activation and the subsequent production of proinflammatory factors. These results suggest that histamine and SP might play an important role in microglial activation and neuroinflammation-related diseases and further our understanding of the regulators and mechanisms involved in the activation of microglia.

The authors declare that there are no conflicts of interest regarding the publication of this paper.

This project was sponsored the National Natural Science Foundation of China (No. 81102422, 81373398); the Natural Science Foundation of Jiangsu Province (BK2010020); and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

J. Zhu and C. Qu contributed equally to this work.