Lipopolysaccharide Priming Exacerbates Anaphylatoxin C5a-Induced Anaphylaxis in Mice

Abstract

Anaphylaxis is a serious allergic or hypersensitivity reaction with a sudden onset that can be life-threatening or fatal. Previous studies have highlighted two pathways of anaphylaxis in mice. One is the classical immunoglobulin E (IgE)-mediated pathway that involves mast cells and histamine. The other is an alternative IgG-mediated pathway that involves basophils, monocytes/macrophages, neutrophils, and the platelet-activating factor (PAF). However, little is known about the mechanism by which complement anaphylatoxins contribute to the induction of anaphylaxis. Infection is a cofactor that potentially amplifies the risk of anaphylaxis. Here, we showed that priming with a lipopolysaccharide (LPS), which mimics bacterial infection, exacerbates anaphylatoxin C5a-induced anaphylaxis in mice. LPS plus C5a-induced anaphylaxis was mediated by histamine and lipid mediators, especially PAF. Cell depletion experiments demonstrated that LPS plus C5a-induced anaphylaxis depended on monocytes/macrophages, basophils, and neutrophils. These results suggest that C5a is a potent inducer of anaphylaxis in bacterial infections. Remarkably, the molecular and cellular mediators of LPS plus C5a-induced anaphylaxis are mostly shared with IgE- and IgG-mediated anaphylaxis. Therefore, combined inhibition of histamine and PAF may be beneficial as a second-line treatment for severe anaphylaxis.

INTRODUCTION

Anaphylaxis is a serious allergic or hypersensitivity reaction with a sudden onset (minutes to a few hours) that can be life-threatening or fatal.^1,2) Because of its life-threatening nature and associated ethical concerns, studies on the underlying mechanisms of anaphylaxis have mainly used mouse models.^3,4) In the classical pathway of anaphylaxis, cross-linking of immunoglobulin E (IgE) on mast cells induces their degranulation with the release of preformed mediators, such as histamine and tryptase, and the production of lipid mediators, such as leukotrienes and prostaglandins, within minutes. In addition to IgE, IgG can induce anaphylaxis. In an alternative pathway, cross-linking of IgG on basophils, monocytes/macrophages, and neutrophils induces their activation with the production of the platelet-activating factor (PAF), another lipid mediator. In most mouse models of anaphylaxis, the combined inhibition of histamine and PAF almost entirely blocks anaphylaxis, suggesting that histamine and PAF are the two major molecular mediators of anaphylaxis. Although the existence of IgG-mediated anaphylaxis in humans remains controversial, a correlation between serum PAF levels and anaphylaxis severity has been reported in human subjects.^5,6)

Complement anaphylatoxins, C3a and C5a, are potent pro-inflammatory peptides generated from C3 and C5 via the activation of C3 and C5 convertases, respectively.⁷⁾ Activation of the canonical complement pathways (classical, alternative, and lectin) converges on C3 convertase, leading to the generation of the anaphylatoxins, C3a and C5a. Anaphylatoxin C5a can also be generated by the activation of non-canonical complement pathways, which are initiated by activated neutrophils, macrophages, platelets, and coagulant cascade proteins, and converge on C5 convertase. Anaphylatoxins target a broad spectrum of immune and non-immune cells.⁸⁾ They activate mast cells and basophils to release histamine and trigger an oxidative burst in macrophages, neutrophils, and eosinophils. Moreover, they regulate vasodilation, increase the permeability of small blood vessels, and induce smooth muscle contractions. Accumulating evidence suggests the possible role of anaphylatoxins in anaphylaxis.^3,4) A correlation between serum anaphylatoxin levels and the severity of anaphylaxis has been reported in humans, although the risk associated with increased anaphylatoxin levels is not as high as that associated with increased tryptase or histamine levels.⁹⁾ In mice, peanut extract activates the complement system via both classical and lectin pathways, resulting in the production of C3a, which contributes to anaphylaxis.^10,11) However, in these studies, mice were pretreated with propranolol (a β-adrenergic antagonist) and a long-acting form of interleukin (IL)-4 to increase the sensitivity to vasoactive mediators. Importantly, peanut extract acts synergistically with IgE-mediated mast cell activation to induce systemic anaphylaxis in the absence of exogenous β-adrenergic antagonists or IL-4.¹⁰⁾ These studies suggest that complement activation can exacerbate anaphylaxis induced by other mechanisms, but may be insufficient to independently induce systemic anaphylaxis.³⁾

Several factors, such as exercise and non-steroidal anti-inflammatory drugs, act as cofactors that amplify the risk of anaphylaxis.^1,2) Infection has been reported as a cofactor in 2.5–3% of anaphylactic episodes in children and 1.3–11% of anaphylactic episodes in adults.¹²⁾ Often, clinicians observe an association between infection and anaphylaxis during allergen immunotherapy. However, the mechanism by which microbial factors amplify the risk of anaphylaxis remains unclear.

We previously reported that stimulation of Ly-6G on neutrophils in lipopolysaccharide (LPS)-pretreated mice induces PAF-mediated lethal anaphylaxis.¹³⁾ This finding led us to speculate that preceding bacterial infection may enhance the ability of innate immune cells to produce molecular mediators of anaphylaxis.

MATERIALS AND METHODS

Mice

Male BALB/c mice (7–12-weeks-of-age) were purchased from Japan SLC (Hamamatsu, Japan) and used in all experiments. Mice were housed under specific pathogen-free conditions. All animal procedures were approved by the Institutional Animal Care and Use Committee of Tohoku University (Sendai, Japan) (2020DnA-038).

Induction of Anaphylaxis by Anaphylatoxin C5a

Room temperature was maintained at 22–23 °C and mice were intravenously injected with recombinant mouse C5a (R&D Systems, Minneapolis, MN, U.S.A.) dissolved in saline (0.1 mL/10 g body weight) at indicated doses. Then, mice were pretreated with an intravenous injection of LPS from Escherichia coli O55:B5 purified via phenol extraction (Sigma-Aldrich, St. Louis, MO, U.S.A.) and dissolved in saline at a dose of 10 μg/kg 2 h before C5a injection. This dose of LPS did not produce any symptoms, and thus, was considered to mimic the subclinical bacterial infection.

Evaluation of the Severity of Systemic Anaphylaxis

Rectal temperature was measured using a digital thermometer (TD-300; Shibaura Electronics, Saitama, Japan) just before C5a injection and at every 10 min for up to 1 h. Mice were euthanized when the rectal temperature fell below 31 °C and/or they appeared moribund (unable to right themselves). The severity of systemic anaphylaxis was evaluated by hypothermia and lethality within 1 h of C5a injection. In some experiments, blood was collected in ethylenediaminetetraacetic acid-containing tubes after C5a injection, and hematocrit was measured using a Celltac hematology analyzer (MEK-5258; Nihon Kohden, Tokyo, Japan). Increase in hematocrit reflected a decrease in plasma volume due to anaphylactic vascular hyperpermeability.

Treatment of Mice

Cromolyn was purchased from Tokyo Kasei Industry (Tokyo, Japan). Pyrilamine, cimetidine, and dexamethasone were purchased from Wako Pure Chemical Corporation (Osaka, Japan). JNJ7777120 and arachidonyl trifluoromethyl ketone (ATK) were purchased from Cayman Chemical (Ann Arbor, MI, U.S.A.). WEB-2086 was purchased from Enzo Life Sciences (Farmingdale, NY, U.S.A.). Control rat IgG was purchased from Rockland Immunochemicals (Pottstown, PA, U.S.A.). RB6-8C5 (anti-Gr-1) antibody was purified from the ascite fluid of hybridoma-bearing mice.¹⁴⁾ Ba103 (anti-CD200R3) antibody was a kind gift from Dr. Hajime Karasuyama (Tokyo Medical and Dental University, Tokyo, Japan).¹⁵⁾ Clodronate liposomes and control phosphate-buffered saline (PBS) liposomes were purchased from Hygieia Bioscience (Osaka, Japan). These reagents were injected into the mice, as described in the Figure legends.

Isolation of Cells from the Lungs and Spleens of Mice

Lungs were cut into small pieces and digested with 50 μg/mL (0.26 units/mL) Liberase TM (Roche, Basel, Switzerland) and 50 μg/mL deoxyribonuclease (DNase) I (Sigma-Aldrich) in RPMI-1640 medium containing 10% fetal bovine serum at 37 °C for 30 min with gentle shaking. After digestion, the lung cells were filtered through a 70-μm nylon mesh. Similarly, the spleen was mashed and filtered through a 70-μm nylon mesh. Lung and spleen cells were hemolyzed with 140 mM ammonium chloride in 17 mM Tris–HCl (pH 7.5). The cells were stained with trypan blue (Wako Pure Chemical Corporation) and viable cells were counted under a microscope using a Neubauer improved hemocytometer (Hirschmann Laborgeräte, Eberstadt Germany). The cells were then subjected to flow cytometry.

Flow Cytometry

The cells were suspended in PBS containing 1% bovine serum albumin and 0.1% sodium azide, and kept on ice throughout the procedure. TruStain FcX (anti-mouse CD16/32) antibody (clone 93; BioLegend, San Diego, CA, U.S.A.) was used to block the Fc receptors. Dead cells were stained with 4′,6-diamidino-2-phenylindole (Dojindo, Kumamoto, Japan). The following antibodies were used: anti-B220 (PE-Cy5, clone RA3-6B2), anti-CD11b (APC-Cy7, clone M1/70), anti-CD45 (BV605, clone 30-F11), anti-CD49b (APC, clone DX5), anti-c-Kit (PE-Cy7, clone 2B8), anti-CX3CR1 (BV421, clone SA011F11), anti-C5a receptor 1 (C5aR1, also known as CD88) (APC, clone 20/70), anti-F4/80 (APC, clone BM8), anti-Ly-6C (PerCP-Cy5.5, clone HK1.4), anti-Siglec-F (Alexa Fluor 488, clone S17007L), anti-TCR (PE-Cy5, clone H57-597) antibodies from BioLegend, along with anti-Ly-6G (BUV737, clone 1A8) antibody from BD Biosciences. Data were acquired using an LSRFortessa cell analyzer (BD Biosciences, San Jose, CA, U.S.A.) at the Biomedical Research Core of the Tohoku University Graduate School of Medicine. The data were analyzed using the FlowJo software version 10.7.1 (Tree Star, Ashland, OR, U.S.A.). The absolute number of each cell type was calculated by multiplying the total number of viable cells isolated from the tissue by the frequency of each cell type.

Statistical Analysis

Statistical analysis was performed using the Prism 9 software (GraphPad), as described in the figure legends. p-Values <0.05 were considered to be statistically significant.

RESULTS

LPS Pretreatment Exacerbates Anaphylatoxin C5a-Induced Anaphylaxis

Single intravenous injections of anaphylatoxin C5a at doses of 20 and 40 μg/kg induced temporal hypothermia, which is indicative of systemic anaphylaxis (Fig. 1A). To test the possible effects of the preceding infection on C5a-induced anaphylaxis, mice were pretreated intravenously with a subclinical dose of LPS (10 μg/kg) 2 h before C5a injection. In LPS-pretreated mice, C5a injections at doses of 10 and 20 μg/kg induced rapid hypothermia and lethal shock within 20 min (Fig. 1B). Moreover, LPS pretreatment augmented the increase in hematocrit, another indicator of systemic anaphylaxis induced by C5a injection (20 μg/kg) (Fig. 1C). These results indicate that LPS pretreatment exacerbates C5a-induced anaphylaxis in mice.

Fig. 1. Lipopolysaccharide (LPS) Pretreatment Exacerbates Anaphylatoxin C5a-Induced Anaphylaxis in Mice

(A) Mice were intravenously injected with C5a at the indicated doses. (B) Mice were intravenously injected with LPS (10 μg/kg). Two hours later, the mice were intravenously injected with C5a at the indicated doses. Rectal temperature was measured at the indicated time-points and expressed as temperature change vs. time 0. Results are represented as the mean ± standard deviation (S.D.); N = 4. † indicates the death of an animal. ** p < 0.01 using two-way repeated measures ANOVA. (C) Mice were intravenously injected with saline or LPS (10 μg/kg), followed by the intravenous injection of saline or C5a (20 μg/kg) after 2 h. Five minutes later, blood was collected to measure hematocrit. Results are represented as the mean ± S.D.; N = 3. ** p < 0.01, *** p < 0.001 using two-way ANOVA with Tukey’s multiple comparisons test. ns, not significant.

LPS Plus C5a-Induced Anaphylaxis Is Mediated by Histamine and Lipid Mediators

To identify the molecular mediators of LPS plus C5a-induced anaphylaxis, the mice were treated with various drugs (Fig. 2). Saline was injected as a control and had no effect on the severity of LPS plus C5a-induced anaphylaxis (Fig. 2A). Anaphylatoxins induce the degranulation of mast cells and basophils to release histamine.⁸⁾ Therefore, inhibition of degranulation by cromolyn partially suppressed the lethality of LPS plus C5a-induced anaphylaxis in this study (Fig. 2B). Among the four histamine receptors (H1R to H4R), the blockade of H1R, H2R, and H4R is beneficial for the treatment of histamine-mediated anaphylactic responses.^16,17) The combined blockade of H1R, H2R, and H4R by anti-histamines (pyrilamine, cimetidine, and JNJ7777120) also partially suppressed the lethality of LPS plus C5a-induced anaphylaxis (Fig. 2C). These results indicate that histamine is a molecular mediator of LPS plus C5a-induced anaphylaxis.

Fig. 2. LPS Plus C5a-Induced Anaphylaxis Depends on Histamine and Lipid Mediators

Mice were intravenously injected with LPS (10 μg/kg), followed by the intravenous injection of C5a (20 μg/kg) after 2 h. (A) Saline (0.1 mL/10 g) was intravenously injected 20 min before C5a injection. (B) Cromolyn (50 mg/kg) was intraperitoneally injected 1 h before C5a injection. (C) A mixture of pyrilamine, cimetidine, and JNJ7777120 (anti-histamines; 10 mg/kg each) was intraperitoneally injected 30 min before C5a injection. (D) Dexamethasone (5 mg/kg) was intraperitoneally injected 2 h before LPS treatment. (E) Arachidonyl trifluoromethyl ketone (ATK; 10 mg/kg) was intraperitoneally injected 1 h before C5a injection. (F) Both anti-histamines and ATK were given as described above. (G) WEB-2086 (3 mg/kg) was intravenously injected 20 min before C5a injection. (H) Both anti-histamines and WEB-2086 were given as described above. Rectal temperature was measured at the indicated time-points and expressed as temperature change vs. time 0. Results are represented as the mean ± S.D.; N = 4. † indicates the death of an animal, the sum of which is shown in parentheses.

Glucocorticoids are often used for the treatment of anaphylaxis owing to their anti-inflammatory effects.^1,2) Dexamethasone, a synthetic glucocorticoid, completely prevented the lethality of LPS plus C5a-induced anaphylaxis (Fig. 2D). Cytosolic phospholipase A2 (cPLA2), which produces arachidonic acid metabolites and PAF, is a key target of glucocorticoids.^18,19) Inhibition of cPLA2 by selective inhibitor ATK only partially prolonged the survival of mice with LPS plus C5a-induced anaphylaxis (Fig. 2E). However, the combined use of anti-histamines and ATK completely suppressed the lethality of LPS plus C5a-induced anaphylaxis (Fig. 2F). Among the lipid mediators generated downstream of cPLA2, PAF is a major mediator of anaphylaxis.^4,19) Although inhibition of the PAF receptor by WEB-2086 was not effective (Fig. 2G), the combined use of anti-histamines and WEB-2086 completely suppressed the lethality of LPS plus C5a-induced anaphylaxis (Fig. 2H). These results indicate that LPS plus C5a-induced anaphylaxis depends on histamine and lipid mediators, particularly PAF.

Monocytes/Macrophages, Basophils, and Neutrophils Are Involved in LPS Plus C5a-Induced Anaphylaxis

Anaphylatoxins stimulate mast cells and basophils to release histamine.⁸⁾ C5aR1 is highly expressed on neutrophils and monocytes/macrophages.⁷⁾ Therefore, these cells are potential cellular mediators of LPS plus C5a-induced anaphylaxis. To identify the specific cellular mediators of LPS plus C5a-induced anaphylaxis, cell depletion experiments were performed (Fig. 3). In separate experiments, depletion of specific cell types in the lungs and spleen by cell-depleting agents was confirmed using flow cytometry (Supplementary Figs. 1–4). The validity of the gating strategy was confirmed by the surface expression of CD49b (a marker for basophils) and F4/80 (a marker for monocytes/macrophages and eosinophils) on gated cells (Supplementary Fig. 5). Injection of control IgG had no effect on the severity of LPS plus C5a-induced anaphylaxis (Fig. 3A). Injection of RB6-8C5 (anti-Gr-1) antibody did not suppress the lethality of LPS plus C5a-induced anaphylaxis (Fig. 3B). Meanwhile, RB6-8C5 depleted neutrophils (defined as CD45⁺CD11b⁺Ly-6G⁺) and Ly-6C^high monocytes (defined as CD45⁺CD11b⁺CX3CR1⁺) (Supplementary Figs. 2, 4). Injection of Ba103 (anti-CD200R3) antibody, which depletes basophils (defined as CD45^lowIgE⁺c-Kit⁻) (Supplementary Fig. 2), partially suppressed the lethality of LPS plus C5a-induced anaphylaxis (Fig. 3C). The control PBS liposomes had no effect on the severity of LPS plus C5a-induced anaphylaxis (Fig. 3D). Injection of clodronate liposomes completely suppressed the lethality of LPS plus C5a-induced anaphylaxis (Fig. 3E). Clodronate liposomes depleted the splenic macrophages (defined as CD45⁺CD11b⁻CX3CR1⁺), but not the alveolar macrophages (defined as CD45⁺Siglec-F⁺CD11b⁻CX3CR1⁺) (Supplementary Figs. 2, 4). Clodronate liposomes also reduced the number of monocytes (Supplementary Figs. 2, 4). These results suggest that LPS plus C5a-induced anaphylaxis depends on monocytes/macrophages.

Fig. 3. LPS Plus C5a-Induced Anaphylaxis Depends on Monocytes/Macrophages, Basophils, and Neutrophils

Mice were intravenously injected with LPS (10 μg/kg), followed by the intravenous injection of C5a (20 μg/kg) after 2 h. (A–E) Control IgG (200 μg/mouse) (A), RB6-8C5 antibody (200 μg/mouse) (B), Ba103 antibody (80 μg/mouse) (C), phosphate-buffered saline (PBS) liposomes (50 μL/mouse) (D), or clodronate liposomes (500 μg/50 μL/mouse) (E) were intravenously injected 24 h before C5a injection. (F, G) Both clodronate liposomes and RB6-8C5 (F) or Ba103 (G) antibody were given as described above. Data are shown in the same way as in Fig. 2. PBS-lip, PBS liposomes; Clo-lip, clodronate liposomes.

Treatment with clodronate liposomes alone was insufficient to suppress the hypothermia induced by LPS plus C5a (Fig. 3E). However, when clodronate liposomes were combined with RB6-8C5 or Ba103, hypothermia was significantly suppressed (Figs. 3F, G). In combination with clodronate liposomes, RB6-8C5 and Ba103 depleted neutrophils and basophils, respectively, in addition to monocytes and splenic macrophages (Supplementary Figs. 2, 4). The combination of clodronate liposomes with RB6-8C5 did not significantly reduce the number of eosinophils (defined as CD45⁺CD11b⁺Siglec-F⁺) although RB6-8C5 alone reduced their number in the lungs (Supplementary Figs. 2, 4). These results suggest that neutrophils and basophils play important roles in LPS plus C5a-induced anaphylaxis.

Recruitment of Neutrophils and Ly-6C^high Monocytes to the Lungs in Response to LPS Injection

During endotoxemia, neutrophils and monocytes are recruited to peripheral tissues, such as the lungs and liver, and contribute to tissue injury by releasing inflammatory mediators.²⁰⁾ Thus, the number of immune cells in the lungs of LPS-treated and untreated mice was determined using flow cytometry (Fig. 4). LPS injection significantly increased the number of neutrophils and Ly-6C^high monocytes, but not other cell types, including Ly-6C^low monocytes and alveolar macrophages, in the lungs. The number of basophils in the lungs was slightly reduced following LPS injection. These results suggest that the priming effects of LPS may be in part due to the recruitment of neutrophils and Ly-6C^high monocytes to the lungs.

Fig. 4. Accumulation of Neutrophils and Ly-6C^high Monocytes in the Lungs after LPS Injection

Lungs were collected from untreated mice and mice that received intravenous injections of LPS (10 μg/kg) 2 h before sacrifice. The cells were isolated and analyzed using flow cytometry. Gating strategy is shown in Supplementary Fig. 1. Absolute numbers of the indicated cell types in the lungs were plotted as the mean ± S.D.; N = 4. * p < 0.05 vs. untreated group using the Mann–Whitney test. ns, not significant.

Rapid Internalization of C5aR1 on Neutrophils and Basophils in Response to C5a Injection

C5aR1 is a classical G-protein-coupled receptor that is rapidly internalized following the binding of its ligand, C5a.⁷⁾ To identify the target cells of C5a, cells isolated from the lungs of mice treated with or without LPS and/or C5a were analyzed using flow cytometry for the surface expression of C5aR1 (Fig. 5; Supplementary Fig. 6). The expression levels of C5aR1 in neutrophils were significantly reduced in C5a-treated mice than in the untreated mice. The expression of C5aR1 in basophils also tended to be reduced by C5a treatment (p = 0.074). Unexpectedly, LPS treatment significantly reduced the expression C5aR1 in the neutrophils. When C5a was administered to LPS-pretreated mice, the expression levels of C5aR1 in neutrophils and basophils were significantly reduced. However, the expression levels of C5aR1 in other cell types were not significantly changed by C5a treatment, with or without LPS pretreatment. Mast cells were excluded from the analysis owing to their rarity. These results suggest that intravenously injected C5a induces anaphylactic responses by acting on neutrophils and basophils.

Fig. 5. Rapid Internalization of C5aR1 on Neutrophils and Basophils in Response to C5a Injection

Lungs were collected from mice: left untreated (labeled as “Untreated”), those who received intravenous injections of C5a (20 μg/kg) 5 min before sacrifice (labeled as “C5a”), those who received intravenous injections of LPS (10 μg/kg) 2 h before sacrifice (labeled as “LPS”), and those who received intravenous injections of C5a (20 μg/kg) 5 min before sacrifice, preceded by the intravenous injection of LPS (10 μg/kg) 2 h before C5a (labeled as “LPS→C5a”). The cells were isolated and analyzed by flow cytometry for surface expression of C5aR1. The experiment was repeated four times with one mouse/group/experiment. Gating strategy and representative histograms are shown in Supplementary Figs. 1 and 6, respectively. The Δ geometric mean fluorescence intensities of C5aR1 in the indicated cell types were plotted. Data from the same experiment were connected by lines. * p < 0.05, ** p < 0.01 using one-way repeated measures ANOVA with Tukey’s multiple comparisons test. GeoMFI, geometric mean fluorescence intensity; ns, not significant.

DISCUSSION

Here, we showed that LPS pretreatment exacerbated anaphylatoxin C5a-induced anaphylaxis in mice. The induction of anaphylaxis by C5a in LPS-pretreated mice was not direct, but rather mediated by histamine and lipid mediators, particularly PAF. Moreover, LPS plus C5a-induced anaphylaxis was found to be dependent on monocytes/macrophages, basophils, and neutrophils. Our results suggest the potential role of C5a in the induction of anaphylaxis in bacterial infections.

Activation of the complement system is tightly regulated, and the plasma concentration of C5a is very low (in the range of 1–5 nM) under normal conditions.⁷⁾ C5a is cleared within 3–5 min via receptor-mediated internalization by immune cells and the removal of carboxyl-terminal arginine residue, which yields less potent C5a-des-Arg.⁷⁾ The normal plasma concentration of C5 (the precursor of C5a) is 0.37 μM.²¹⁾ Under certain disease conditions, the plasma concentration of C5a can exceed 100 nM.⁷⁾ In the present study, intravenous injection of 10 and 20 μg/kg of C5a induced lethal anaphylaxis in LPS-pretreated mice. Assuming the molecular mass of recombinant mouse C5a to be 9.0 kDa (according to the datasheet) and the plasma volume of male mice to be 56 mL/kg,²²⁾ plasma concentration of the injected C5a (10 μg/kg) was calculated to be approximately 20 nM. Our results suggest that clinically relevant doses of C5a can induce lethal anaphylaxis if the individuals are primed by inflammatory stimuli, such as bacterial infections.

We demonstrated that LPS plus C5a-induced anaphylaxis depends on histamine and lipid mediators, particularly PAF. These are known molecular mediators of IgE- and IgG-mediated anaphylaxis.^3,4) Thus, it is likely that under the present experimental conditions, C5a exerted its anaphylaxis-inducing effects not by acting directly on endothelial or smooth muscle cells, but rather through the release of histamine and lipid mediators from immune cells. This is consistent with previous studies showing that cardiac dysfunction caused by C5a is mediated by histamine, adenosine, and lipid mediators, such as thromboxane A₂ and leukotrienes.²³⁾ Peanut extract can contribute to anaphylaxis by producing C3a, which stimulates immune cells to produce PAF and histamine.¹⁰⁾ We previously reported that anaphylaxis induced by LPS plus stimulation of Ly-6G on neutrophils is mediated by PAF.¹³⁾ These results suggest that the principal molecular mediators are shared in anaphylaxis induced by different mechanisms. If so, the combined inhibition of histamine and cPLA₂ metabolites, especially PAF, may be beneficial as a second-line treatment for most cases of anaphylaxis. Although anti-histamines are used for the treatment of anaphylaxis, no drugs targeting PAF are currently available.^1,2)

Results of cell depletion experiments suggest that the LPS plus C5a-induced anaphylaxis depends on monocytes/macrophages, basophils, and neutrophils. All these cell types expressed C5aR1 on their surfaces, as demonstrated using flow cytometry. C5aR1 belongs to the superfamily of G protein-coupled receptors and couples primarily to the Gαi subtype of heterotrimeric G proteins.²⁴⁾ C5aR2, also known as C5L2 or GPR77, is another receptor for C5a, the function of which is controversial. In contrast to C5aR1, C5aR2 lacks functional coupling to heterotrimeric G proteins and its expression is predominantly intracellular.²⁴⁾ Thus, in this study, we focused on C5aR1. C5aR1 is rapidly internalized upon binding of C5a, and can serve as a marker for C5a exposure.^7,25) The surface expression levels of C5aR1 on neutrophils and basophils were reduced 5 min after the intravenous injection of C5a in both LPS-pretreated and untreated mice, indicating that C5a activates neutrophils and basophils to release the molecular mediators of anaphylaxis. Such a significant reduction in the surface expression of C5aR1 after the intravenous injection of C5a was not observed in monocytes/macrophages and eosinophils. The surface expression of C5aR1 in the neutrophils was downregulated by LPS, implying their exposure to C5a in the LPS-induced priming phase. LPS can lead to C5a generation through the alternative complement pathway as well as through the activation of macrophages (a non-canonical complement pathway).^7,26) However, it is unclear if subclinical dose of LPS used in this study involves C5a generation. In the context of sepsis, downregulation of surface C5aR1 expression in neutrophils has been associated with their dysfunction and poor outcomes in patients.⁷⁾ Thus, it is important to examine the generation of C5a and the active status of neutrophils in the context of subclinical endotoxemia in future studies.

Activation of neutrophils requires an initial priming step to prevent their unregulated activation.²⁷⁾ Neutrophils can be primed via contact with the activated endothelium, foreign surfaces, or agents, such as LPS. Priming not only enhances the activation potential of neutrophils, but also recruits neutrophils to the site of inflammation, such as the lungs. Similar priming effects have been reported for Ly-6C^high monocytes.²⁸⁾ Consistently, we observed the recruitment of neutrophils and Ly-6C^high monocytes to the lungs during the LPS-induced priming phase. However, depletion of both neutrophils and Ly-6C^high monocytes by the RB6-8C5 antibody alone did not prevent the lethality of LPS plus C5a-induced anaphylaxis, suggesting that other cell types may play redundant roles in this process. LPS pretreatment upregulated the surface expression levels of CD14, a co-receptor for LPS with Toll-like receptor 4 and MD-2, in neutrophils, Ly-6C^high and Ly-6C^low monocytes, and macrophages (data not shown). Thus, we assume that Ly-6C^low monocytes and macrophages are also primed by LPS to respond to secondary stimuli in a manner that does not involve the recruitment of cells. Considering that the depletion of monocytes/macrophages by clodronate liposomes alone completely prevented the lethality of LPS plus C5a-induced anaphylaxis, monocytes/macrophages may play an important role in the LPS-induced priming phase. The number of basophils was slightly reduced in the lungs after LPS pretreatment. In addition, basophils expressed little surface CD14 and its expression was not upregulated by LPS pretreatment (data not shown). These results suggest that LPS pretreatment has no priming effect on basophils.

One limitation of this study is that we could not identify the cellular sources of molecular mediators. As discussed above, our results suggest that basophils and neutrophils are direct targets of C5a, and that neutrophils and monocytes/macrophages are primed by LPS. Both basophils and neutrophils produce histamine and PAF.^4,29) Thus, it is likely that C5a activates basophils and neutrophils to release histamine and PAF in LPS-pretreated mice. LPS induces histamine release by neutrophils and macrophages^29,30) and increases the ability of neutrophils to produce PAF.¹³⁾ Various cell types, including neutrophils and macrophages, produce lipid mediators in response to PAF.³¹⁾ LPS primes macrophages to enhance PAF-induced PAF production.³²⁾ Therefore, LPS pretreatment enhances the ability of neutrophils and monocytes/macrophages to produce histamine and PAF, and C5a-elicited PAF stimulates further production of lipid mediators from neutrophils and monocytes/macrophages.

In conclusion, we found that a subclinical dose of LPS exacerbated anaphylatoxin C5a-induced anaphylaxis, which was mediated by histamine and lipid mediators, particularly PAF, produced by monocytes/macrophages, basophils, and neutrophils. This study supports the notion that infection works as a cofactor of anaphylaxis, and that C5a is a potent inducer of anaphylaxis. Remarkably, the above-mentioned molecular and cellular mediators are mostly shared with IgE- and IgG-mediated anaphylaxis processes. Therefore, combined inhibition of histamine and PAF may potentially be used as a universal second-line treatment for severe anaphylaxis.

Acknowledgments

This work was supported by the Japan Society for the Promotion of Science (Grant Nos. 22K10164 to MY and 24890022 to YT).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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