Platelets Control Leukocyte Recruitment in a Murine Model of Cutaneous Arthus Reaction

Platelets have been shown to be important in inflammation, but their role in the cutaneous Arthus reaction remains unclear. To assess the role of platelets in this pathogenetic process, the cutaneous Arthus reaction was examined in wild-type mice and mice lacking E-selectin, P-selectin, or P-selectin glycoprotein ligand-1 (PSGL-1) with or without platelet depletion by busulfan, a bone marrow precursor cell-specific toxin. Edema and hemorrhage induced by immune complex challenge significantly decreased in busulfan-treated wild-type mice compared with untreated mice. Busulfan treatment did not affect edema and hemorrhage in P-selectin- or PSGL-1-deficient mice, suggesting that the effect by busulfan is dependent on P-selectin and PSGL-1 expression. The inhibited edema and hemorrhage paralleled reduced infiltration of neutrophils and mast cells and reduced levels of circulating platelets. Increased cutaneous production of interleukin-6, tumor necrosis factor-α, and platelet-derived chemokines during Arthus reaction was inhibited in busulfan-treated wild-type mice relative to untreated mice, which paralleled the reduction in cutaneous inflammation. Flow cytometric analysis showed that immune complex challenge generated blood platelet-leukocyte aggregates that decreased by busulfan treatment. In thrombocytopenic mice, the cutaneous inflammation after immune complex challenge was restored by platelet infusion. These results suggest that platelets induce leukocyte recruitment into skin by forming platelet-leukocyte aggregates and secreting chemokines at inflamed sites, mainly through the interaction of P-selectin on platelets with PSGL-1 on leukocytes.The pathogenesis of autoimmune diseases frequently involves the formation of IgG-containing immune complexes (ICs) inducing inflammatory responses with significant tissue injury, commonly referred to as type III hypersensitivity reaction. This IC injury has been implicated in the pathogenesis of vasculitis syndrome, systemic lupus erythematosus, rheumatoid arthritis, and cryoglobulinemia.¹ The mechanisms by which the immune system controls effector responses to ICs are of central importance for developing therapeutic strategies. The standard animal model for the inflammatory response in these IC-mediated diseases is the Arthus reaction.² Analyses using gene knockout mice have revealed that activation of the complement system, especially C5a and its interaction with C5a receptor, and of Fc receptors for IgG on inflammatory cells, particularly mast cells, are both required to initiate the Arthus reaction.^3–8 In addition, accumulation of neutrophils and mast cells is necessary for the progression of the IC-mediated vascular tissue damage, which results in edema and hemorrhage.^3–8Leukocyte recruitment from the circulation to a site of inflammation is an essential process in the inflammatory response. Leukocytes first tether and roll on vascular endothelial cells, before they are activated to adhere firmly and subsequently immigrate into the extravascular space. This multistep process is highly regulated by multiple cell-surface adhesion molecules.^9,10 The selectins cooperate to support leukocyte tethering and rolling along inflamed vascular walls by mediating leukocyte interactions with glycoconjugated counter-receptors expressed by endothelium, adherent platelets, or leukocytes. The selectin family consists of three cell-surface molecules expressed by leukocytes (L-selectin), vascular endothelium (E- and P-selectins), and platelets (P-selectin).¹¹ Although the adhesive mechanisms underlying the capture and immobilization of circulating leukocytes in inflamed blood vessels have been well described, factors triggering and controlling the leukocyte recruitment into inflamed sites are poorly understood.The multistep process of leukocyte tethering and rolling, followed by leukocyte activation and firm adhesion, also occurs on activated platelets.¹² Platelets are essential for primary hemostasis, but they also play an important pro-inflammatory role.^13,14 Platelets normally circulate in a quiescent state, protected from untimely activation by inhibitory mediators released from intact endothelial cells. Endothelial dysfunction and changes in release of antiplatelet factors lead to increased platelet activation followed by their interaction with leukocytes, and increased platelet adhesion and aggregation.^15,16 On activation, platelets can change their shapes as well as the expression pattern of adhesion molecules, and secrete neutrophil and endothelial activators inducing production of pro-inflammatory cytokines.¹⁷ These changes are associated with the adhesion of platelets to leukocytes and endothelium.¹⁴ Thus, platelets are important amplifiers of acute inflammation.Platelets accumulate in inflammatory lesions concomitantly with leukocytes and regulate a variety of inflammatory responses by secreting or activating adhesion proteins, growth factors, and coagulation factors.^18,19 These proteins induce widely differing biological activities, including cell adhesion, chemotaxis, cell survival, and proliferation, all of which accelerate the inflammatory process.²⁰ In vitro and in vivo studies have shown that platelets bind to leukocytes through their surface protein.^12,14,20,21 Indeed, previous studies have reported that platelet-leukocyte aggregates are formed in circulating blood of asthmatic patients.²² Platelets express much amounts of P-selectin than endothelium and also bind endothelium via selectin dependent and independent mechanisms.^23–25 In addition to classical leukocyte recruitment process, platelets bound to activated endothelial cells can interact with leukocytes, which results in secondary capture that induces interactions of leukocytes with platelets first, followed by leukocyte-endothelial cell interaction.²⁶ Leukocytes within platelet-leukocyte complexes have increased adhesive capacity to the activated endothelium.²⁷ Therefore, platelet can function as a bridge between the circulating leukocyte and endothelium.We previously showed that mice lacking P-selectin (P-selectin^−/−) or mice treated with anti- P-selectin glycoprotein ligand-1 (PSGL-1) antibody (Ab) exhibited reduced Arthus reaction that is associated with decreased infiltration of neutrophils and mast cells.^28,29 In addition to interacting with selectins and selectin ligands on endothelial cells, leukocytes can also interact with selectins and selectin ligands presented by platelets or their microparticle fragments, which are all found at sites of inflammation.³⁰ This indicates that observations of altered leukocyte recruitment in selectin- and selectin ligand-deficient mice must be discussed in light of altered selectin and selectin-ligand expression not only by endothelial cells, but also by platelets. Recently, involvement of platelets has been demonstrated in the pathogenesis of inflammatory disorders, including asthma,^22,31 arthritis,¹⁸ inflammatory bowel disease,³² and chronic allergic dermatitis.³³ Although the role of platelets in inflammatory process is being increasingly recognized, it remains unknown how platelets induce leukocyte recruitment in the cutaneous Arthus reaction. A recent report has identified a role of platelets in promoting IC-induced leukocyte recruitment to the cremaster muscle in a murine model of reverse passive Arthus reaction.³⁴ However, the relative role of each leukocyte and adhesion molecule in the inflammation varies according to the tissue site and the nature of inflammatory stimuli.²⁹ Therefore, to clarify the importance of platelets, their surface adhesion molecule expression, and platelet-derived chemokines on leukocyte recruitment, we examined the cutaneous Arthus reaction in wild-type, P-selectin^−/−, E-selectin^−/−, and PSGL-1^−/− mice, with or without treatment with busulfan, a bone marrow precursor cell-specific toxin.