Introduction

Allergic diseases, including asthma and allergic rhinitis have become a significant global health concern, affecting hundreds of millions of individuals worldwide1,2. These conditions are characterized by aberrant immune responses to a variety of allergens and environmental insults, leading to airway inflammation and tissue remodelling3,4,5. Asthma and chronic rhinosinusitis are characterized by disruption of the epithelial barrier in the lower and upper airways due to various triggers (e.g., allergens, smoke, microbial products, pollutants)4,6. Damaged epithelial cells rapidly release several cytokines, including thymic stromal lymphopoietin (TSLP), IL-33, and IL-25 collectively known as “alarmins”7,8. These cytokines activate a wide spectrum of cells in both the innate and adaptive immune system, including lung mast cells9,10,11,12 and macrophages13,14.

Histamine is a proinflammatory and vasoactive mediator stored in the cytoplasmic granules of mast cells and basophils15, playing multiple roles in the pathobiology of allergic disorders16,17. Mast cells and basophils activated by IgE-mediated (i.e., allergens in sensitized individuals or anti-IgE autoantibodies)18,19,20 and non-IgE-mediated stimuli9,21 release histamine, leading to a cascade of inflammatory responses that trigger the characteristic symptoms of allergic diseases (e.g., itching, sneezing, and bronchoconstriction)17,22,23. Histamine exerts its effects on immune cells through its interaction with four known histamine receptors: H1, H2, H3, and H424. These receptors are heptahelical transmembrane G protein-coupled receptors (GPCRs) differently expressed on a variety of immune cells, including mast cells17, eosinophils25, basophils26, T and B cells27,28, monocytes29, dendritic cells (DCs)30, and macrophages29. Activation of histamine receptors on immune cells can modulate cytokine production and immune responses, thereby contributing to the pathobiology of allergic disorders25,29.

The prevailing narrative in allergic disease research has long emphasized the pivotal role of type 2 inflammation in the pathobiology of asthma and other allergic disorders31. In this context, IgE-mediated mast cell activation and eosinophilic inflammation play key effector roles32,33,34. Although these cells undoubtedly play crucial roles as effector cells, the role of lung macrophages has often been overshadowed. Macrophages represent the most abundant immune cells in human lung35,36 and play complex roles in allergic diseases37 as well as in different phenotypes of asthma38,39. There is now increasing evidence that both tissue-resident and recruited lung macrophages are key orchestrators of chronic airway inflammation through different mechanisms40,41,42.

Mast cells are strategically located in different lung compartments of healthy subjects and patients with asthma37. These cells reside in close proximity to macrophages in the human lung14,43,44. Activated lung mast cells secrete histamine and this release is increased in asthmatic patients45. Previous studies have evaluated the effects of histamine on human peripheral blood mononuclear cells46 and monocyte-derived DCs30. By contrast, the potential impact of histamine on human lung macrophages (HLMs) has been subject of limited studies, which suggested that these cells express the HRH147,48.

In this study, we investigated the effects of histamine on various aspects of the activation of macrophages highly purified from human lung49. In particular, we evaluated the in vitro effects of histamine on the release of several proinflammatory and immunomodulatory cytokines, the production of reactive oxygen species (ROS), autophagy, and the kinetic properties of HLMs.

Methods

Reagents

The following reagents were purchased: histamine, bafilomycin A1 [Sigma-Aldrich (St. Louis, US)], cetirizine [Thermo Fisher Scientific (Waltham, MA, USA)] L-glutamine, fetal bovine serum (FBS), lipopolysaccharide (LPS) (from Escherichia coli serotype 026:B6), Percoll®, piperazine- N, N’-bis (2-ethanesulfonic acid) (PIPES), phosphate buffer saline (PBS), Triton X-100, antibiotic–antimycotic solution (10,000 IU/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin B) (Lonza, Basel, CH), RPMI 1640 (Microgem, Naples, Italy), DMSO (Merck Millipore, Burlington, MA, USA), 2′,7′-dichlorofluorescin diacetate (H2DCFDA) (Thermo Fisher Scientific, Waltham, MA, USA). Target-specific primers for HRH1, IL-6, TNF-α, IL-1β, and GAPDH were designed using the Beacon Designer 3.0 (Bio-Rad Laboratories, Milan, Italy) and produced and purified by Custom Primers (Life Technologies, Milan, Italy).

Isolation and purification of human lung macrophages (HLMs)

Macrophages were purified from macroscopically normal lung tissue of 15 patients (aged 45–79 years, hepatitis C virus−, hepatitis B surface Ag−, HIV1−) undergoing lung resection at the Istituto Nazionale Tumori IRCCS Fondazione G. Pascale. The study protocol was approved by the Comitato Etico Territoriale Campania 1 (Prot. 36/23 OSS) and informed consent was obtained from patients undergoing thoracic surgery. Lung tissue was minced finely with scissors, washed with piperazine-N, N′-bis (2-ethanesulfonic acid (PIPES) buffer over Nitex cloth (120 μm pore size; Sefar Italia), and the dispersed cells recovered. The macrophage suspension was enriched (75–85%) by flotation over discontinuous Percoll® density gradients13,14. The cells were suspended (106 cells/mL) in complete medium (RPMI 1640 supplemented with 5% FCS, 2 mM L-glutamine, 1% antibiotic–antimycotic solution, and 1% non-essential amino acids) and incubated in 24-well plates at 37 °C. After 18 h, the medium was removed, and the plates were gently washed with fresh medium. More than 98% of adherent cells were macrophages (HLMs), as evaluated by flow-cytometric analysis, as previously demonstrated49. HLMs used in Fig. 1 were obtained from 6 donors (age range: 45–79 years, mean age: 68 years, 4 males, 2 female). HLMs used in Figs. 3, 4 and 5, and 6 included 3 of the same donors as in Fig. 1 (age range: 52–75 years, mean age: 64 years, 2 male, 1 female). HLMs used in Fig. 2 were obtained from 3 different donors, not overlapping with those used in Fig. 1 or Figs. 3, 4, 5 and 6 (age range: 50–79 years, mean age: 56 years, 1 male, 2 female). HLMs used in Fig. 7 were obtained from 3 different donors, not overlapping with those used in any other figure (age range: 60–72 years, mean age: 66 years, 2 males, 1 female). HLMs used in Fig. 8 were obtained from 3 different donors, not overlapping with those used in any other figure (age range: 45–70 years, mean age: 62 years, 1 male, 2 female).

Fig. 1
figure 1

Effects of histamine on cytokine release from human lung macrophages (HLMs). HLMs (106 cells/well) were incubated (18 h at 37 °C) with complete medium (CTR), increasing concentrations of histamine (10−6−10−4 M) or LPS (1 µg/mL). IL-6 (A), TNF-α (B), and IL-1β (C) concentrations in supernatants were evaluated by ELISA and expressed as ng of mediators for mg of total proteins. Data are the mean ± SD of six independent experiments obtained from different donors. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. CTR.

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Fig. 2
figure 2

Effects of histamine on mRNA expression for cytokines in human lung macrophages (HLMs). HLMs (106 cells/well) were incubated (3 to 18 h at 37 °C) with complete medium (CTR) or histamine (10−4 M). At the end of incubations, IL-6 (A), TNF-α (B), and IL-1β (C) mRNAs were determined by quantitative RT-PCR. Data are mean ± SD of three independent experiments obtained from different donors. * p < 0.05 vs. CTR.

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Histamine stimulation of HLMs

Histamine was diluted in fresh RPMI 1640 medium supplemented with 5% FBS immediately before each experiment. HLMs were cultured in 24-well plates (0.15-1 × 106 HLMs/well) in complete medium and then stimulated (18 h at 37 °C) with increasing concentrations of histamine (10−6- 10−4 M) or with LPS (1 µg/mL). In another series of experiment, HLMs preincubated (30 min at 37 °C) with increasing concentrations of cetirizine (10−7- 10−5 M) and were then incubated (18 h at 37 °C) with increasing concentrations of histamine (3 × 10−6- 3 × 10−3 M). At the end of incubations, supernatants were harvested, centrifuged (500 × g, 10 min at 4 °C), and stored at − 80 °C for subsequent determination of mediator release. Lysis of the cell pellets in the plates was carried out using 0.1% Triton X-100. Total protein quantification was performed by a Bradford assay (Bio-Rad Laboratories, Segrate MI, Italy).

Cell viability

Cell viability was evaluated as mitochondrial activity, determined by the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay, as previously reported50. HLMs (106 HLMs/well) were incubated with histamine (10−6 – 10−4 M), 1% v/v Triton X-100, or medium alone for 18 h at 37 °C. At the end of incubations, supernatants were removed and the cells were incubated (1 h at 37 °C) in 1 mL of MTT solution (0.5 mg/mL). The cells were washed with PBS, 0.5 mL of DMSO was added, and absorbance was read at 540 nm. Cell injury was expressed as a percentage relative of cultures incubated with medium alone.

ELISA assays

The release of soluble mediators in the supernatants of HLMs was measured in duplicate samples using commercially available ELISA kits for IL-6, TNF-α, IL-1β, CXCL1, CXCL2, CXCL8, vascular endothelial growth factor (VEGF)-A, angiopoietin 1 (ANGPT1), and ANGPT2 (R&D Systems, Minneapolis, MN, USA). Since the number of HLMs can vary among wells and different experiments, the results were normalized for the total protein content in each well, determined in the cell lysates (0.1% Triton X-100). Therefore, all mediator values were expressed as ng or pg/mg of total proteins.

RT-PCR

IL-6, TNF-α, IL-1β, HRH1, and p62 mRNA expressions were evaluated in HLMs. Cells (106 cells/well) were incubated (3, 6, and 18 h at 37 °C) with histamine (10−4 M). After stimulation, supernatants were removed and HLMs were lysed to evaluate mRNA expression. Total RNA was extracted by TRIzol® reagent (Euroclone, Milan, Italy) following the manufacturer’s instructions. RNA quality and integrity were estimated by spectrophotometric analysis using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Real-time RT-PCR was performed using Universal SYBR Green Supermix (Bio-Rad Laboratories, Segrate, MI, Italy) utilizing a CFX96 real-time detection system (Bio-Rad Laboratories, Segrate, MI, Italy). GAPDH was used as a housekeeping gene to normalize cycle threshold (Ct) values using the 2−ΔCt formula35. PCR efficiency and specificity were evaluated by analyzing amplification curves with serial dilutions of the template cDNA and their dissociation curves. Each cDNA sample was analyzed in triplicate and the corresponding no-RT mRNA sample was included as a negative control. The data were analyzed with iCycleriQ analysis software (Bio-Rad Laboratories, Segrate, MI, Italy) and the changes in IL-6, TNF-α, IL-1β, HRH1, and p62 mRNAs were expressed as 2−ΔCt.

Reactive oxygen species (ROS) production

HLMs (1.5 × 105 cells/well) were seeded in a 96-well for 30 min at 37 °C after the addition of 10 µg/mL 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) (Life Technologies, Milan, Italy)51. H2DCF-DA is a fluorogenic dye that allows the evaluation of hydroxyl, peroxyl and other ROS activities within the cell. Once diffused into the cell, H2DCF is deacetylated by cellular esterases to a nonfluorescent molecule, which is oxidized by ROS into 2′,7′-DCF. This latter compound is highly fluorescent and can be determined by fluorescence spectroscopy with maximum excitation and emission wavelengths of 492–495 nm and 517–527 nm, respectively. Cells were washed in PBS, then resuspended in RPMI 1640 supplemented with 5% of FBS and finally seeded in a 96-well pre-coated plate with medium alone or increasing concentrations of histamine. Immediately after stimulation, the plate was placed in an EnSpire Multimode Plate Reader (Perkin Elmer, Waltham, MA, USA). The ability of stimuli to induce cytoplasmic ROS-catalyzed oxidation of H2DCF in HLMs was compared to the negative control (medium alone). The data were expressed as 2′,7′-DCF relative fluorescence units (RFU), normalized to T0, and measured up to 30 min with 2 min span51.

Western blotting of autophagic markers

HLMs were lysed in an ice-cold buffer containing 100 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM EGTA, 1 mM phenylmethylsulphonyl fluoride, 1 mM Na3VO4, 1 mM NaF, 0.5% NONIDET P-40, 0.2% SDS, and a protease inhibitor mixture (Roche Diagnostics, Indianapolis, IN, USA). After centrifugation (13,000 rpm, 30 min at 4 °C), the supernatants were collected. Protein concentration of each sample was calculated by the Bradford assay. Protein samples (20 µg) were separated on 15% sodium dodecyl sulfate-polyacrylamide gels and then electrotransferred onto 0.2 μm nitrocellulose membranes (GE Healthcare, Milan, Italy). Then, membranes were incubated with the following primary antibodies: anti-p62 (NBP1-48320, Novus Biologicals, Littleton, CO, USA) (1:1,000), anti-LC3B (GTX127375, GeneTex Inc., Irvine, CA, USA) (1:1,000), and anti-β-actin peroxidase (A3854, Sigma-Aldrich, Milan, Italy) (1:10,000)52.

Chemotaxis assay

Migration of HLMs toward histamine was evaluated using 8 μm cell culture inserts in 96-well companion plates (Sigma-Aldrich, Milan, Italy)53. The companion plates were loaded with 235 µl of RMPI 1640 serum-free (without FBS), increasing concentrations of histamine (dissolved in RPMI 1640 serum-free), and FBS (as positive control). HLMs (1.8 × 105 macrophages/75 µl) were placed in the inserts and allowed to migrate for 1 h at 37 °C in the presence of 5% CO2. At the end of the incubations, the cells were centrifuged and resuspended in 100 µl of PBS and counted by flow cytometry (MACSQuant Analyzer 10, Miltenyi Biotec, Germany)53.

Time-lapse and high-content microscopy

Time-lapse and high-content microscopy experiments were conducted with the Operetta High-Content Imaging System (PerkinElmer, MA, USA), as previously described54,55. HLMs were cultured in Falcon® 24-well Clear Flat Bottom plates. For time-lapse experiments, HLMs were incubated with histamine (10−6 M) or complete medium for 48 h at 37 °C. Within this time window, digital phase contrast images of 15 fields/well were captured every hour via a 10× objective. To quantify cell tracking features, bright-field snapshots were taken at 6 fields/well. PhenoLOGIC (PerkinElmer, Milan, Italy) was also employed to analyze kinetic proprieties as current displacement X, displacement X mean per well, current displacement Y, displacement Y mean per well, current speed, and speed mean per well55.

Statistical analysis

Statistical analysis was performed using Prism 9 (GraphPad Software, San Diego, CA, USA). The data are expressed as mean values ± standard deviation (SD) of the indicated number of experiments. Data were compared by Student’s t-test or one-way analysis of variance (ANOVA) followed by Dunnett’s test (when comparison was made against a control) or Bonferroni’s test (when comparison was made between each pair of groups) by means of Analyse-it for Microsoft Excel, version 2.16 (Analyse-it Software, Ltd., Leeds, UK). P < 0.05 was considered statistically significant.

Results

Differential effects of histamine on the release of proinflammatory mediators from HLMs

In a series of experiments, we evaluated the effects of increasing concentrations of histamine (10−6−10−4 M) on the activation of highly purified HLMs from six different donors. Histamine concentration-dependently activated HLMs to release IL-6 (Fig. 1A), TNF-α (Fig. 1B), and IL-1β (Fig. 1C). The activation effects of histamine on cytokine release were significant at 10−4 M. In these experiments, we used LPS, the main component in the cell wall of Gram-negative bacteria, as a positive control56,57. The same concentrations of histamine did not affect the release of several chemokines (CXCL1, CXCL2, CXCL8) and different angiogenic factors (VEGF-A, ANGPT1, ANGPT2)58,59 (Supplementary Fig. 1). The percentage of viable HLMs, measured by MTT assay 18 h after histamine treatment, did not differ from that of untreated cells (Supplementary Fig. 2 A). To exclude the possibility that the activating effects of histamine could be due to marginal LPS contamination, HLMs were incubated with histamine alone or in the presence of polymyxin B (50 µg/mL), a potent inactivator of LPS60. Polymyxin B did not modify the spontaneous release of IL-6, TNF-α, and IL-1β (Supplementary Fig. 2B-D). Moreover, incubation of histamine with polymyxin B did not alter the activating property of histamine (10−4 M) on the release of cytokines from HLMs. As expected, polymyxin B significantly reduced the release of tested mediators induced by LPS from HLMs (Supplementary Fig. 2B-D).

We then investigated whether histamine induced mRNA expression for cytokines (IL-6, TNF-α, IL-1β) in HLMs using real-time PCR. In these experiments, we used the optimal concentration of histamine (10−4 M) for different times (3, 6, and 18 h), which induced the transcription of mRNA for IL-6 (Fig. 2A), as previously reported47. By contrast, histamine did not induce the de novo production of both TNF-α (Fig. 2B), and IL-1β (Fig. 2C), at any of these time points. These results support the hypothesis that histamine induced the release of preformed TNF-α and IL-1β from HLMs, but not their de novo synthesis.

Effect of cetirizine on histamine-induced cytokine-release from HLMs

Histamine exerts its effects primarily by binding to G protein-coupled receptors (GPCRs), designated histamine receptors H1 through H4 (HRH1/2/3/4)24. Among them, HRH1 is the major receptor involved in allergic responses and HRH1 antagonists (e.g., cetirizine) are widely used to relieve allergy symptoms61. In a series of experiments, we evaluated whether histamine-induced cytokine release was due to HRH1 activation. HLMs were preincubated (30 min at 37 °C) with increasing concentration of cetirizine (10−7−10−5 M), a specific HRH1 antagonist61,62, and then stimulated with histamine (3 × 10−6−3 × 10−3 M). Figure 3A and B show the results of typical experiments out of three. Increasing concentrations of cetirizine caused a parallel shift to the right of the dose-response curve induced by histamine on the release of TNF-α (Fig. 3A) and IL-1β (Fig. 3B). Experiments such as those shown in Fig. 3A and C were used to construct Schild plot analysis63. The intercept on the abscissa provided the dissociation constant (KD) of the cetirizine-HRH1 complex on HLMs. Figure 3B and D show the results obtained in three independent experiments, which indicated that the KD of cetririzine-HRH1 complex was (mean ± SD) 7.65 × 10−8 ± 7.84 × 10−9 M for TNF-α and 8.14 × 10−8 ± 1.30 10−8 M for IL-1β. These results are compatible with the observation that cetirizine is a competitive inhibitor at the level of HRH164.

Fig. 3
figure 3

Effect of varying concentrations of histamine, alone and in combination with cetirizine, on cytokine release from human lung macrophages (HLMs) (A, C). HLMs were preincubated (30 min at 37 °C) with increasing concentrations of cetirizine (10−7- 10−5 M) and then incubated (18 h at 37 °C) with varying concentrations of histamine (3 × 10−6- 3 × 10−3 M). TNF-α and IL-1β concentrations in supernatants were evaluated by ELISA and expressed as ng of mediators for mg of total proteins. (B-D) In three independent experiments, the concentration of histamine, alone and in combination with cetirizine, needed to produce 50% of maximal effect was calculated. For each concentration of cetirizine, the dose ratio was calculated63. The KD was obtained from the value of log (cetirizine) where log (dose-ratio-1) = 063. The different symbols (●, ○, ◻) refer to the results from three experiments obtained from different donors.

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Effects of histamine on ROS production from HLMs

Reactive oxygen species (ROS) play an essential role in macrophage activation and polarization51. Considering the key role of oxidative stress and angiogenesis65 in allergic inflammation, we evaluated whether histamine could activate ROS production in HLMs. Increasing concentrations (10−7- 10−4 M) of histamine-induced ROS production in time-dependent manner from HLMs. Interestingly, different from cytokine production, the most effective concentration of histamine in ROS production was 10−6 M (Fig. 4A-B). The rapidity of ROS production technically precluded the evaluation of an HRH1 antagonist on the release of this mediator from HLMs (Cristinziano et al.., unpublished observations).

Fig. 4
figure 4

Effect of histamine on reactive oxygen species (ROS) production from human lung macrophages (HLMs). (A, B) 2′,7′-dichlorodihydrofluorescein diacetate-labeled HLMs were incubated (30 min at 37 °C) with complete medium (CTR) or increasing concentrations of histamine (10−7−10−5 M). Fluorescence was measured for 30 min at 2-min intervals. Data are normalized on T0 and are mean ± SD of three independent experiments with cells from different donors. ** p < 0.01 vs. CTR.

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Effects of histamine on autophagy in HLMs

The intricate interplay between ROS production, autophagy, and asthma pathobiology66 stimulated our interest to investigate the effect of histamine on autophagy in HLMs. Autophagy serves as a cleaning mechanism to remove noxious agents and it contributes to the development and functions of several immune cells including macrophages67,68. Autophagy is primarily a tissue-protective mechanism that exerts anti-inflammatory effects68,69. In a series of three experiments, we evaluated the effects of histamine (10−6 M) on different markers (i.e., LC3-I, LC3-II and their ratio, and p62) of autophagy70 in HLMs52. Figure 5A shows the results of a typical experiment indicating that histamine increased protein expression in HLMs. Moreover, histamine increased p62, a different marker of autophagic pathway52,70. Figure 5B and C, and 5D show the results of three experiments demonstrating that histamine significantly increased protein expression of LC3-I and LC3-II and their ratio, and of p62. Uncropped gels of Western blot of Fig. 5 are reported in Supplementary Fig. 3. This could be interpreted as an accumulation of autophagic substrates due to autophagy dysfunction. Therefore, to better assess the impact of histamine on autophagic flux, we conducted Western blotting experiments to detect LC3 and p62 in HLM treated with either vehicle or bafilomycin A1, a blocker of autolysosome formation. Histamine induced an accumulation of LC3-II, detected as LC3-II/Actin ratio, and p62, measured as p62/Actin ratio, that were further increased by the addition of bafilomycin A1 (100 nM and 1 µM/1 hour) (Supplementary Fig. 4B-C). This suggests a degradation of autophagosomes and functional autophagy in HLM exposed to histamine. Concomitantly, histamine did not change p62 transcripts when added for 6 and 18 h to HLMs (Supplementary Fig. 4 A). On the other hand, p62 mRNA was significantly enhanced after 3 h of exposure (data not shown). This suggests an initial failure of autophagosomes degradation but functional autophagy in HLM exposed to histamine. Moreover, under control conditions, HLMs were capable of modulating autophagic flux potently blocked by bafilomycin A1 that produced a significant increase in LC3-II and p62 protein expression. Collectively, these data suggested that histamine partially reduced autophagic flux in HLMs that could be useful to handle some, but yet unexplored, HLM functions.

Fig. 5
figure 5

Effects of histamine on p62, LC3-I and LC3-II protein expression in human lung macrophages (HLMs). HLMs were incubated (18 h at 37 °C) in the presence of control medium (CTR) or histamine (10−6 M). (A) Representative Western blot of p62, LC3-I, LC3-II and β−actin. The simultaneous overexpression of both p62, a substrate of autophagy degradation, and LC3-II by histamine indicates its ability to induce accumulation of these autophagy markers as sign of autophagy blockade in HLMs. Densitometric analyses of p62 (B), LC3-II (C), and LC3-II/LC3-I ratio (D) compared to β–actin expression. Data are presented as means ± SD of three independent experiments obtained from different donors. * p < 0.05 vs. CTR.

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Effects of histamine on HLM chemotaxis

Chemotaxis plays a crucial role in the pathobiology of allergic diseases by mediating the recruitment of immune cells to sites of inflammation, contributing to tissue damage71. In contrast to most leukocytes, macrophages as archetypes of tissue-resident immune cells, were considered to lack motility within the tissues72. By contrast, recent evidence demonstrates that the majority of lung macrophages can critically move in response to chemotactic stimuli73. In a series of three experiments, we evaluated the effects of increasing concentrations of histamine (10−6−10−4 M) on HLM chemotaxis by flow cytometry. In these experiments, FBS was used as positive control. The effect of histamine on HLM chemotaxis was significant at 10−4 M compared to the control medium measured both as cell count (Fig. 6A) and percentage of migrated cells by histamine vs. unstimulated cells (Fig. 6B). As expected, FBS also induced chemotaxis of HLMs.

Fig. 6
figure 6

Effect of histamine on chemotaxis of human lung macrophages (HLMs). HLMs (1.8 × 105 cells/mL in 75 µL) were allowed to migrate (60 min at 37 °C) toward complete medium (CTR), increasing concentrations of histamine (10−6 −10−4 M) or FBS (235 µL per well). At the end of incubation, the cells were centrifuged and resuspended in phosphate-buffered saline (PBS) (100 µL) and counted by flow cytometry. Graphs show raw data of HLMs (counts/mL) (A) and migratory cells normalized for control medium (B). Data are the mean ± SD of three independent experiments obtained from different donors. * p < 0.05 vs. CTR.

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Effects of histamine on kinetic properties of HLMs

Different stimuli can change immune cell morphology as well as their movement74. We evaluated HLM tracking induced by histamine with an Operetta High-Content Imaging System54,55. HLMs were incubated with histamine (10−6 M) or complete medium for 48 h at 37 °C. Figure 7 illustrates the results of three independent experiments showing the effects of histamine on the kinetic proprieties of HLMs. When the starting point was set to 0 on the X-axis and 0 on the Y-axis, histamine induced significant changes in cell displacement along X-axis (Fig. 7A) and Y-axis (Fig. 7C). When we analyzed the results from three experiments, histamine significantly reduced the displacement X mean per well (Fig. 7B) and Y mean per well (Fig. 7D) compared to unstimulated cells. Histamine had no effect on the speed of HLMs (Fig. 7E and F).

Fig. 7
figure 7

Effects of histamine on kinetic properties of human lung macrophages (HLMs). HLMs (105 cells/well) were incubated (48 h at 37 °C) with complete medium (CTR) or histamine (10−6 M). The incubation was carried out with time-lapse and high-content microscopy Operetta High-Content Imaging System to investigate the tracking characteristics as current displacement X (A), displacement X mean per well (B), current displacement Y (C), displacement Y mean per well (D), current speed (E), and speed mean per well (F). Data in Fig. 7B and D, and 7E are the mean ± SD of three independent experiments obtained from different donors. ** p < 0.01; * p < 0.05 vs. CTR.

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Effect of histamine on the expression of HRH1 receptor on HLMs

The above results demonstrate that histamine can induce a variety of biological effects presumably through the activation of HRH1 on HLMs. We have previously demonstrated that HLMs express HRH129. Therefore, we investigated whether histamine (10−4 M) can influence HRH1 mRNA expression in HLMs. Figure 8 shows the results from three independent experiments demonstrating that histamine induced the upregulation of HRH1 mRNA in HLMs.

Fig. 8
figure 8

Effect of histamine on the expression of H1 receptor (HRH1) mRNA in human lung macrophages (HLMs). HLMs (106 cells/well) were incubated (6 hours at 37 °C) with complete medium (CTR) or histamine (10−4 M). At the end of incubation, HLMs were lysed and RNA was extracted. mRNA expression for HRH1 was evaluated by quantitative RT-PCR. The red dotted line represents the control values. Data are the mean ± SD of three independent experiments obtained from different donors. * p < 0.05 vs. CTR.

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Discussion

Our study provides novel findings demonstrating that histamine induces the concentration-dependent release of cytokines (IL-6, TNF-α, IL-1β) from highly purified HLMs. Interestingly, histamine induced IL-6 mRNA, but not for TNF-α and IL-1β. These effects were mediated by the activation of HRH1, as they were blocked by cetirizine, a second-generation antihistamine62,75. Moreover, histamine upregulated HRH1 in HLMs. Low concentrations of histamine induced ROS production and increased specific markers of autophagy. Finally, histamine induced HLM chemotaxis and affected macrophage tracking.

Histamine, released from activated mast cells and basophils9,59,76, is canonically considered a typical proinflammatory and vasoactive mediator77,78. Increasing evidences demonstrate that this biogenic amine exerts extensive effects on several relevant immunologic cell types, including mast cells17, eosinophils25, basophils26, T and B cells27,28, monocytes29,47, and DCs30. Histamine can exert its pleiotropic effects on immune cells through the activation of four (HRH1, HRH2, HRH3, and HRH4) receptors24. Our results extend our previous observation29 that histamine can cause the release of IL-6 from HLMs, the predominant immune cells in the lung of healthy and asthmatic subjects36. In this study, we found that histamine can induce the release of IL-6 protein and the mRNA expression of this cytokine. By contrast, histamine induced the release of two important cytokines, IL-1β and TNF-α, without stimulating their mRNA expression. Collectively, these results emphasize a dual mechanism of action of histamine on the release of cytokines from HLMs.

Another interesting peculiarity of the effects of histamine is due to the lack of stimulating effect on the release of two different classes of proinflammatory and immunomodulating mediators. In fact, histamine did not modify the release of several chemokines (CXCL1, CXCL2, and CXCL8) and angiogenic factors (VEGF-A, ANGPT1, and ANGPT2) from HLMs. The latter findings highlight the complexity and specificity of the activating properties of histamine on HLMs. Although 10⁻⁴ M histamine exceeds typical circulating plasma levels, local histamine concentrations in inflammatory/allergic tissue microenvironments can transiently reach high micromolar to submillimolar peaks upon mast cell/basophil degranulation79. Several in vitro studies have employed comparable concentrations to probe histamine-mediated signaling and mediator release in immune cells46,80.

Histamine can activate a variety of immune cells through the engagement of HRH127,47,81, HRH227,81,82,83,84, HRH385, and HRH486,87,88,89,90,91. In our experiments, the histamine-induced release of IL-6, TNF, and IL-1beta was antagonized by the second-generation antihistamine cetirizine62. Cetirizine appears to be a competitive antagonist at the HRH1 receptor, inhibiting both the de novo synthesis of IL-6 and the exocytotic release of TNF-α and IL-1β induced by histamine. The specificity of these findings is supported by the fact that the KD, the dissociation constant of the inhibitory effect of cetirizine, is essentially the same in both processes of histamine-induced cytokine release from HLMs.

ROS play a critical role in several aspects of airway remodeling92,93,94, angiogenesis65, macrophage activation and polarization51. We found that histamine rapidly (10 to 30 min) increased ROS production from HLMs. The interplay between ROS, autophagy and asthma pathobiology66 prompted us to evaluate the effect of histamine on autophagy biomarkers in HLMs. Increasing evidence suggests that macrophages are one of the bridges connecting autophagy and immunity68,95. We found that histamine increased the protein expression of two major markers of autophagy (i.e., P62, LC3-II). These results support the hypothesis that histamine may cause a defective autophagic flux without ultimately limiting the process in HLMs. The partial reduction of autophagy flux could be useful to temporally handle different immunological functions that are unknown. To our knowledge, this is the first evidence suggesting that histamine is a possible modulator of autophagy in HLMs. Histamine concentration required to induce significant ROS production and autophagy modulation (10−6 M) was lower than that needed to elicit substantial cytokine release (10−4 M). This difference in sensitivity could be attributed to the heterogeneity of lung macrophages37,42,96. It is conceivable that distinct subpopulations possess varying levels of HRH1 expression or downstream signaling components, leading to differential responsiveness to histamine.

Chemotaxis and the kinetic properties of immune cells play a fundamental role in the pathobiology of several immune disorders71. This aspect is particularly relevant in the lungs, which are continuously exposed to various environmental insults97 and microbial products98. Although the chemotaxis of most leukocytes is crucial in immune defense and pathobiology of inflammatory disorders, macrophages are considered archetypes of tissue-resident immune cells72. We found that histamine promoted the chemotaxis of HLMs. Our in vitro findings agree with an elegant in vivo demonstration that lung macrophages in mice can move providing efficient immune surveillance73. Moreover, using an Operetta High-Content Imaging System, we found that histamine significantly affected the kinetic properties of HLMs. This histamine-induced modulation of macrophage tracking may have important biological and clinical implications. By migrating to specific locations within the airway wall, histamine-stimulated macrophages can release high local concentrations of IL-6, TNF-α, and IL-1β, amplifying the proinflammatory cytokine milieu and promoting recruitment of other immune cells. Furthermore, macrophages positioned at sites of epithelial injury or allergen deposition may drive extracellular matrix deposition and airway smooth muscle cell proliferation, contributing to the airway remodelling characteristic of chronic asthma99.

Collectively, the above results demonstrate that histamine can influence a variety of biological responses in HLMs. Some of these effects are mediated by the engagement of HRH1 in HLMs, and histamine can upregulate HRH1 in these cells. The latter observations have translational relevance, considering the close proximity between mast cells and macrophages within human lung parenchyma43,44. Lung mast cells, particularly those of allergic subjects45, release histamine, which upregulates HRH1 on macrophages thus amplifying this autocrine circuit at the site of inflammation. Based on these findings, it is possible that the beneficial effects of HRH1 antagonists may be mediated, at least in part, by the inhibition of histamine-induced cytokine release and ROS production from HLMs.

We cannot exclude the possibility that our findings might also have some relevance in the field of cancer. Mast cells are widely present in almost all human and mouse cancers100,101. Moreover, elevated levels of histamine have been detected in cancer patients’ blood and cancerous tissues102–104. It has been recently reported that taking HRH1-antihistamines correlated with better survival of cancer patients105. Interestingly, HRH1 expression was positively correlated with tumour-associated macrophages (TAMs) in the tumour microenvironment, particularly in immunosuppressive M2-like macrophages. Incidentally, our population of purified HLMs express the M2 phenotype49. Finally, an HRH1 antagonist (fexofenadine) restored T cell antitumor immunity105. The above results agree with our findings showing that histamine induced the release of proinflammatory cytokines (i.e., IL-6, TNF-α, IL-1β) and ROS, involved in tumor initiation and progression106,107. Further research is needed to investigate the involvement of histamine as a mediator of cross-talk between TAMs and mast cells in cancer.

This study has certain limitations that should be pointed out. Our results were obtained using highly purified HLMs. However, lung macrophages are heterogenous37,42,96 and we cannot exclude the possibility that histamine exerts different effects on distinct subpopulations of HLMs. To fully elucidate the subpopulation-specific effects of histamine and the underlying mechanisms, future studies employing single-cell RNA sequencing (scRNA-seq) are warranted. In some experiments, we used relatively high concentrations of histamine. It is possible that, within human lung, activated mast cells, particularly those from allergic individuals45, release high local concentrations of histamine similar to those used in our in vitro experiments. Our experiments were performed using primary macrophages obtained from macroscopically normal lung parenchyma of patients undergoing thoracic surgery for lung cancer. We cannot exclude the possibility that the underlying disease may have influenced some of our findings.

Conclusion

Primary human lung macrophages express the HRH1 which can be upregulated by histamine. Histamine induced the release of several proinflammatory and immunoregulatory cytokines (IL-6, IL-1β, TNF-α) through different mechanism involving the activation of HRH1. Although histamine did not induce the release of several angiogenic mediators and chemokines, it promoted the rapid formation of ROS. Finally, histamine stimulated chemotaxis and affected the kinetic properties of HLMs. Collectively, our results indicate that histamine, as a mediator of cross-talk between human mast cells and macrophages, could be involved in chronic inflammatory lung disorders.