RhoA signaling through platelet P2Y1 receptor controls leukocyte recruitment in allergic mice

doi:10.1016/j.jaci.2014.09.032

Journal of Allergy and Clinical Immunology

Volume 135, Issue 2, February 2015, Pages 528-538.e4

https://doi.org/10.1016/j.jaci.2014.09.032 Get rights and content

Background

Clinical studies reveal platelet activation in patients with asthma, allergic rhinitis, and eczema. This is distinct from platelet aggregation, which is critical for the maintenance of hemostasis and in which a role for platelet purinergic receptors is well documented. However, purines are also essential for inflammatory cell trafficking in animal models of allergic lung inflammation, which are known to be platelet dependent, yet the role of purines in the platelet activation accompanying inflammation is unknown.

Objectives

We investigated whether the involvement of purine activation of platelets during allergic inflammation is distinct from purine involvement in platelet aggregation.

Methods

BALB/c mice were sensitized to ovalbumin and subsequent airway ovalbumin challenge. Bronchoalveolar lavage fluid was analyzed for inflammatory cells, and blood samples were assessed for platelet activation. The role of platelet purinergic receptors and associated signaling mechanisms (RhoA) were assessed.

Results

P2Y₁, but not P2Y₁₂ or P2X₁, antagonism inhibited pulmonary leukocyte recruitment. The formation of platelet-leukocyte complexes in vivo and platelet/P-selectin–dependent polymorphonuclear cell migration in vitro were exclusively platelet P2Y₁ receptor dependent. Furthermore, platelet P2Y₁ activation resulted in RhoA activity in vivo after allergen challenge, and RhoA signaling in platelets through P2Y₁ stimulation was required for platelet-dependent leukocyte chemotaxis in vitro. Leukocyte recruitment in thrombocytopenic mice remained suppressed after reinfusion of platelets pretreated with a P2Y₁ antagonist or a Rho-associated kinase 1 inhibitor, confirming the crucial role of platelet P2Y₁ receptor and subsequent activation of RhoA.

Conclusion

RhoA signaling downstream of platelet P2Y₁, but not P2Y₁₂, represents a clear dichotomy in platelet activation during allergic inflammation versus hemostasis.

Section snippets

Methods

Reagents used and details of the receptor pharmacology for the purinergic antagonists used in this study can be found in the Methods section in this article's Online Repository at www.jacionline.org. The methods outlined below appear in the order in which the experiments are described in the Results section.

ADP activation of P2Y₁, but not P2Y₁₂, is critical for pulmonary leukocyte recruitment in a murine model of allergic inflammation

Purines have been reported to induce the expression of selectins and integrins on the surfaces of platelets and leukocytes32, 34, 35 and are necessary for leukocyte chemotaxis.41, 42 Therefore it is feasible that purines are involved in the pulmonary recruitment of leukocytes during allergic inflammation.28, 37 OVA-sensitized mice were administered apyrase (100 U/mL) before each allergen challenge to test this hypothesis, which significantly attenuated pulmonary eosinophil, neutrophil,

Discussion

We provide evidence of a distinct mechanism of platelet activation in the context of allergic inflammation leading to pulmonary leukocyte recruitment. Specifically, costimulation of the platelet P2Y₁ receptor by ADP is required, leading to downstream RhoA activation (Fig 8). This represents a divergence of platelet activation by inflammatory compared with aggregatory stimuli, in which ADP, acting to costimulate platelets, does not lead to platelet aggregation. Significantly, others have shown

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Neutrophils, the early responders of the immune system, eliminate intruders, but their over-activation can also instigate tissue damage leading to various autoimmune and inflammatory disease conditions. As approaches causing neutropenia are associated with immunodeficiency, targeting aberrant neutrophil infiltration offers an attractive strategy in neutrophil-centered diseases including acute lung injury. Rho GTPase family proteins Rho, Rac and Cdc42 play important role as regulators of chemotaxis in diverse systems. Rho inhibitors protected against lung injuries, while genetic Rho-deficiency exhibited neutrophil hyperactivity and exacerbated lung injury. These differential outcomes might be due to distinct effects on different cell types or activation/ inhibition of specific signaling pathways responsible for neutrophil polarity, migration and functions. In this study, we explored neutrophil centric effects of Rho signaling mitigation. Consistent with previous reports, Rho signaling inhibitor Y-27632 provided protection against acute lung injury, but without regulating LPS mediated systemic increase of neutrophils in the circulation. Interestingly, the adoptive transfer approach identified a specific defect in neutrophil migration capacity after Rho signaling mitigation. These defects were associated with loss of polarity and altered actin dynamics identified using time-lapse in vitro studies. Further analysis revealed a rescue of stimulation-dependent L-selectin shedding on neutrophils with Rho signaling inhibitor. Surprisingly, functional blocking of L-selectin (CD62L) led to defective recruitment of neutrophils into inflamed lungs. Further, single-cell level analyses identified MAPK signaling as downstream mechanism of Rho signaling and L-selectin mediated effects. p-AKT levels were diminished in detergent resistance membrane-associated signalosome upon Rho signaling inhibition and blockade of selectin. Moreover, inhibition of AKT signaling as well as selectin blocking led to defects in neutrophil polarity. Together, this study identified Rho-dependent distinct L-selectin and AKT signaling mediated regulation of neutrophil recruitment to inflamed lung tissue.
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Clinical evidence exists demonstrating changes in platelet behavior and function in inflammatory diseases and infections of the respiratory tract, in particular in asthma, rhinitis, acute lung injury arising from Adult Respiratory Distress Syndrome (ARDS), COVID-19 and chronic obstructive pulmonary disease (COPD). Despite being anucleate, platelets participate in the pathogenesis of these disorders in a manner distinct from their effects in thrombosis and hemostasis. These include the expression of adhesion molecules, the release of proinflammatory mediators, tissue-degrading enzymes, cationic proteins, and the ability to undergo chemotaxis. Platelets can themselves be activated by mediators released by other inflammatory cells. There is now substantial experimental evidence that supports a role for platelets in the development of immunity, leukocyte recruitment to inflamed tissue, airways hyperresponsiveness, lung remodeling and repair, and pulmonary hypertension.
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Pulmonary fibrosis is a progressive chronic inflammatory lung disease whose pathogenesis is complicated. Platelets and neutrophils play important roles in the progression of pulmonary inflammation. We have reported that cangrelor, a non-sepesific GPR17 antagonist, alleviates pulmonary fibrosis partly by inhibiting macrophage inflammation in mice. Cangrelor is also a well-known anti-platelet agent. To test whether cangrelor mitigated pulmonary fibrosis partly through the inhibition of platelets, bleomycin (BLM) was used to induce pulmonary fibrosis in C57BL/6 J mice. We found that cangrelor (10 mg/kg) not only significantly decreased BLM-induced release of inflammatory cytokines (PF4, CD40 L and MPO), but also decreased the increment of platelets, neutrophils and platelet-neutrophil aggregates in the fibrotic lung and in the peripheral blood of BLM-treated mice. In addition, cangrelor decreased the number of CD40 and MPO double positive neutrophils and the expression level of CD40 in BLM-treated mouse lungs. Based on these results we conclude that cangrelor alleviates BLM-induced lung inflammation and pulmonary fibrosis in mice, partly through inhibition of platelet activation, therefore reducing the infiltration of neutrophils due to the adhesion of platelets and neutrophils mediated by CD40 - CD40 L interaction. Cangrelor could be a potential therapeutic medicine for pulmonary fibrosis.
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Platelets are under investigation for their role in host defence and inflammatory lung diseases and have been demonstrated to be recruited to the lung. However, the mechanisms and consequences of platelet recruitment into lungs are poorly understood. We have utilised a murine model to investigate the mechanisms of platelet involvement in lung inflammation induced by intranasal administration of LPS.
Our aim was to characterise lung platelet recruitment following LPS inhalation in mice using immunohistochemistry, and non-invasive and invasive radiolabelled platelet tracking techniques.
Intranasal administration of LPS caused an increase in lung platelet staining in lung tissue and elicited the recruitment of radiolabelled platelets into the lung. Prior to these responses in the lung, we observed an earlier decrease in blood platelet counts, temporally associated with platelet recruitment to the liver and spleen. Non-invasive measurements of thoracic radioactivity reflected changes in blood counts rather than extravascular lung platelet recruitment. However, both in situ counting of radiolabelled platelets and immunostaining for platelet surface markers showed LPS-induced increases in extravascular platelets into lung airspaces suggesting that some of the platelets recruited to the lung enter air spaces.
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: Supported by the BBSRC (grant no. BB/H015728/1) and the Sackler Foundation and a grant from the Italian Ministry of University and Research (MIUR prot. 2012773NE3; to P.G.).

: Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest.

View full text

Mechanisms of allergy and clinical immunologyRhoA signaling through platelet P2Y1 receptor controls leukocyte recruitment in allergic mice

Background

Objectives

Methods

Results

Conclusion

Section snippets

Methods

ADP activation of P2Y1, but not P2Y12, is critical for pulmonary leukocyte recruitment in a murine model of allergic inflammation

Discussion

Lancet

J Allergy Clin Immunol

J Allergy Clin Immunol

J Allergy Clin Immunol

J Allergy Clin Immunol

Blood

Blood

J Biol Chem

Blood

J Thromb Haemost

Biophys J

Blood

Blood

Immunol Lett

Trends Pharmacol Sci

Platelet activation and allergic asthma

N Engl J Med

Production of serotonin by tryptophan hydroxylase 1 and release via platelets contribute to allergic airway inflammation

Am J Respir Crit Care Med

Platelet activation during antigen-induced airway reactions in asthmatic subjects

N Engl J Med

Platelet activation in allergic asthma patients during allergen challenge with Dermatophagoides pteronyssinus

Clin Exp Allergy

Increased releasability of platelet products and reduced heparin-induced platelet factor 4 release from endothelial cells in bronchial asthma

J Lipid Mediat

Markers of platelet activation in plasma of patients suffering from persistent allergic rhinitis with or without asthma symptoms

Clin Exp Allergy

Platelet activation in the lung after antigen challenge in a model of allergic asthma

Am Rev Respir Dis

Platelet dynamics following allergen challenge in allergic asthmatics

Respiration

Increased circulating platelet microparticles as a potential biomarker in asthma

Allergy

Platelets in bronchoalveolar lavage from asthmatic patients and allergic rabbits with allergen-induced late phase responses

Agents Actions Suppl

Bronchial biopsies in asthma. An ultrastructural, quantitative study and correlation with hyperreactivity

Am Rev Respir Dis

Platelet activation, P-selectin, and eosinophil β1-integrin activation in asthma

Am J Respir Crit Care Med

Allergen induces the migration of platelets to lung tissue in allergic asthma

Am J Respir Crit Care Med

Mechanisms of allergy and clinical immunology
RhoA signaling through platelet P2Y₁ receptor controls leukocyte recruitment in allergic mice

ADP activation of P2Y₁, but not P2Y₁₂, is critical for pulmonary leukocyte recruitment in a murine model of allergic inflammation