钟文德 张媛莉
（广东医科大学附属医院重症医学科；广东湛江524000）
【摘要】 血小板与巨噬细胞均在免疫及炎症反应中扮演重要角色，而且两者间存在相互作用，可通过直接和（或）间接接触的协同作用影响炎症的结局。深入探究其三者间的关系，有利于更深入的了解炎症的发生发展过程，可能为治疗炎症特别是脓毒症提供一种新依据和新策略。本文主要综述血小板与巨噬细胞在免疫及炎症方面的作用，并探讨二者间相互关系对炎症发生发展的影响。
【关键词】血小板；巨噬细胞；炎症；脓毒症
炎症是机体对损伤因子所做出的一种防御反应，适度的炎症反应有利，过度的炎症反应则会损伤机体，产生严重后果甚至死亡，例如脓毒症的发生。脓毒症是机体对感染反应失调导致危及生命的器官功能障碍，其发病率及死亡率一直居高不下[1]，主要特点是非特异性免疫功能障碍和免疫失衡，是感染、创伤、休克等急危重症患者的严重并发症[2]。血小板是先天免疫反应的重要组成部分，对有害信号进行监测和快速反应[3]，并且在炎症反应、病原体杀灭和组织修复中发挥重要作用[4-7]。巨噬细胞也是先天免疫的重要组成部分，通过调节炎症反应和吞噬功能，在维持细胞稳态和宿主细胞防御系统中发挥重要作用[8]。因此，血小板和巨噬细胞均在炎症中扮演重要角色，而其两者之间直接接触和（或）间接接触的协同运作方式也对炎症产生复杂影响。本文扼要概述血小板及巨噬细胞在免疫方面的作用及其二者间相互关系对炎症发生发展的影响。
一、血小板
血小板是骨髓窦血管中多倍体巨核细胞碎裂的过程中释放到血流中大小2-4μm、无核的、盘状的细胞质碎片[9]。在人体内，血小板的供应和清除受机体稳定的调控，其数量在血液中维持在1.5-4.0×10^5/ul。血小板的生成非常依赖于血小板生成素(TPO)，而TPO在巨核祖细胞增殖/分化和巨核细胞成熟过程中起着重要作用[10]。血小板的寿命很短，最长可达10天。它们通过脾、肝巨噬细胞和肝细胞识别凝集素-碳水化合物的机制被清除[11, 12]。除骨髓不断规律生成血小板外，肺生成的血小板约占血小板总量的50%[13]。血小板内包含3种颗粒，包括α颗粒、致密颗粒和溶酶体[14]。其中，α颗粒是迄今为止最丰富的，每个血小板中含50~80个，其次是致密颗粒（3~6个），溶酶体最少（0~3个）[15]。而α颗粒含有可溶性P-选择素、可溶性CD40L、血小板因子4（platelet factor 4）、RANTES（CCL5）、IL-1β和IL-1α等多种细胞因子，可上调白细胞，特别是单核细胞和巨噬细胞的促炎功能[16]。表明血小板在免疫反应方面具有重要作用。
二、巨噬细胞
巨噬细胞是非常重要的免疫细胞，在体内广泛存在。巨噬细胞是高度可塑性的细胞，可以根据各种信号改变极化[17]。根据周围环境的不同，巨噬细胞可以采用不同的功能表型，主要包括经典激活表型(M1)和交替激活表型(M2)。M1型主要是表达促炎因子，如IL-6/8/1β、TNF-α、ROS和RNS等，而M2型主要参与组织重塑和免疫调节，分泌CD163、CD206等；分泌抗炎细胞因子，如IL-10[18]。微生物刺激、组织损伤以及组织微环境改变均可影响巨噬细胞极化表型，体现了巨噬细胞极化在空间及时间上的可塑性，而这种可塑性对于炎症反应的启动和消退至关重要。
三、血小板与巨噬细胞的直接接触
血小板与巨噬细胞的直接接触作用体现在两者细胞膜表面信号传递的接触，引发下游信号通路的激活。血小板表面表达TOLL样受体-4（TLR-4）[19]、糖蛋白Ib（GPIb）[20]、P-选择素[21]、Fc受体[22]等，巨噬细胞膜表面表达CD11b[23]，CD206，巨噬细胞抗原-1（Mac-1）[24]、清道夫受体（CD163）等。其中血小板GPIb扮演重要角色。血小板 GPIb 受体是一种异质二聚体，由GPIbα和 GPIbβ组成，在血小板膜上与GPV和GPIX形成GPIb-V-IX复合物，介导血小板与内皮细胞黏附。血小板GPIbα与整合素Mac-1结合，通过叉头转录因子Foxp1协调单核细胞分化为巨噬细胞[25]；血小板可以通过GPIb和肝窦Kupffer细胞上表达的von Willebrand因子之间的“接触和移动”机制包裹细菌[26]。Carestia等[27]发现血小板GPIb与巨噬细胞CD11b以GPIb-CD11b轴介导巨噬细胞往M1型巨噬细胞极化，阻断GPIb或CD11b均不能使巨噬细胞往M1极化；且血小板参与巨噬细胞早期极化编程，GPIb-CD11b细胞轴可能在此扮演重要角色。
血小板减少症通常被定义为血小板计数<150*109/L,脓毒症相关性血小板减少症（SAT）是重症监护病房(ICU)最常见的止血疾病，患病率约为50%[28]，SAT发病机理复杂，单核/巨噬细胞清除[29]是其中重要机制之一，单核细胞和巨噬细胞表达Fc-γ受体移除，该受体可以与血小板结合抗体的Fc部分相互作用，从而吞噬血小板。GPIb在脓毒症相关性血小板减少症中也发挥重要作用，一项体外研究发现LPS诱导的脓毒症小鼠模型中，通过腹腔注射抗GPIbα单抗可加剧血小板减少，从而导致器官衰竭加重，增加小鼠死亡率[30]。此外，Rong等[31]发现在免疫性血小板减少症（ITP）患者中，血小板GPIb-α与肝巨噬细胞整合素αIIbβ3作用可导致血小板聚集、吞噬和快速清除，导致血小板减少。血小板GPIb与巨噬细胞之间的关系是治疗SAT的潜在靶点。
四、血小板与巨噬细胞的间接接触
血小板颗粒介质的释放是血小板功能的核心[32]，越来越多的研究表明，血小板不需要与巨噬细胞直接接触就能影响其功能[33]。它们的间接作用主要体现在血小板分泌的介质对巨噬细胞功能形态的调控从而影响炎症的发生发展。
1、PAF
血小板激活因子(PAF)是一种强效的磷脂介质，除血小板分泌外，单核/巨噬细胞、中性粒细胞等细胞接受炎症信号刺激后也可产生，介导血小板聚集、过敏反应和炎症[34]。PAF被认为是急性胰腺炎[35]、糖尿病[36]和肾功能衰竭[37]等炎性疾病的有效介导物。PAF可增加肺泡巨噬细胞的吞噬能力和产生超氧阴离子[38]。此外，在免疫防御过程中，PAF对单核/巨噬细胞也有免疫调节作用，包括单核/巨噬细胞脱颗粒、细胞因子释放、吞噬和细胞黏附[39, 40]。
在体外，PAF已被证明能使巨噬细胞“启动”或重新编程，从而对随后的内毒素刺激产生增强的细胞因子反应[41]。McManus等人[42]发现PAF受体拮抗剂治疗可减轻LPS诱导的肺损伤。巨噬细胞炎性蛋白-2(MIP-2)是巨噬细胞和上皮细胞分泌的一种强有力的中性粒细胞趋化因子，在血小板激活因子(PAF)作用下，可募集中性粒细胞释放炎症因子，介导肠道炎症和损伤，是脓毒症引起急性肠损伤的重要发病机制之一[43]。
2、CCL5
CCL5(RANTES)是血小板中最常见的趋化因子之一，血小板是CCL5的主要来源[44]，是一种抑制巨噬细胞炎症的细胞因子[45]，属于CC趋化因子家族。Rundk等[46]发现血小板Rho家族Rac1调节CCL5的分泌，CCL5作用肺泡巨噬细胞上的CC趋化因子受体5（CCR5），促进肺泡巨噬细胞分泌CXC趋化因子2（CXCL2）有效地诱导中性粒细胞募集，导致脓毒症肺损伤。Aswad等[47]发现CCL5也可发挥抗炎作用，在炎症消退期，CCL5可与巨噬细胞上一种非典型趋化因子受体D6结合，使巨噬细胞重编程，巨噬细胞促炎因子和趋化因子的释放减少，伴随着TGF-β和IL-10的增加[48-50]，促进凋亡细胞分解和伤口修复。
3、β2M
血小板可以表达主要组织相容性复合体I类(MHC I)[51]，MHC I由MHCα链和β2M组成。β2M是MHCα细胞表面转运的伴侣，β2M在膜上稳定MHCα。血小板是血浆β2m的主要来源，β2M除了其分子伴侣作用外，还具有重要的免疫调节功能[52]。Zachary等[53]发现PLT-β2M-/来源的单核细胞则呈现“促修复”的表型，血小板衍生的β2M对单核细胞具有直接的促炎作用，使单核细胞分化为M1型巨噬细胞，血小板通过β2M调节单核细胞表型维持炎症信号和修复信号之间的平衡，这可能成为脓毒症中血小板调节单核细胞分化和巨噬细胞极化的另一重要病理生理机制。
4、PF4
血小板因子4(PF4)是一种血小板特异性肝素结合蛋白，在活化血小板α颗粒中含量丰富[54]，归类为CXC趋化因子家族，也被称为CXCL4。PF4趋化作用较弱，促炎作用相对较强[55]。PF4是一种单核细胞存活因子，可诱导单核细胞分化为巨噬细胞[56]，诱导巨噬细胞吞噬能力增强[57]。在细菌脓毒症中，PF4与需氧细菌表面的糖胺聚糖(GAGs)结合，形成抗原复合物，可能早期诱导产生抗GAGs/PF4 IgA-IgG-IgM[58, 59]作为一种防御机制，促进吞噬、先天免疫[60]和自身免疫[61]，而慢性革兰氏阴性感染患者中也发现了这些抗体[62]。 Ley等[63]发现PF4也可调节单核细胞分化为巨噬细胞，且巨噬细胞表型表现为缺乏CD163、不同于“M1”和“M2”的M4型巨噬细胞，介导组织纤维化。
五、结语及展望
血小板与巨噬细胞在免疫及炎症方面均有重要作用，且二者间关系紧密。血小板是巨噬细胞极化重塑的一个强有力的调控因素，而巨噬细胞直接参与到血小板减少症中，是SAT的重要机制。血小板可以通过直接或间接接触的方式调控巨噬细胞的功能状态，影响巨噬细胞在炎症中发挥的作用，从而影响炎症的结局。研究探索血小板与巨噬细胞相互关系在炎症中的作用有利于更深入的了解炎症，或能为炎症特别是脓毒症治疗提供新依据和新策略。
【参考文献】
[1] M S, CS D, CW S, et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3).[J]. JAMA, 2016,315(8):801-810.
[2] 薄禄龙, 卞金俊, 邓小明. 2016年脓毒症最新定义与诊断标准：回归本质 重新出发[J]. 中华麻醉学杂志, 2016,36(03):259-262.
[3] Vardon-Bounes F, Ruiz S, Gratacap M P, et al. Platelets Are Critical Key Players in Sepsis[J]. Int J Mol Sci, 2019,20(14).
[4] Riedemann N C, Guo R F, Ward P A. The enigma of sepsis[J]. J Clin Invest, 2003,112(4):460-467.
[5] MR T, RF S. The role of platelets in inflammation.[J]. Thrombosis and haemostasis, 2015,114(3):449-458.
[6] Pierrakos C, Vincent J. Sepsis biomarkers: a review[J]. Critical care (London, England), 2010,14(1):R15.
[7] Aziz M, Jacob A, Yang W L, et al. Current trends in inflammatory and immunomodulatory mediators in sepsis[J]. J Leukoc Biol, 2013,93(3):329-342.
[8] Ali M, Bonay M, Vanhee V, et al. Comparative effectiveness of 4 natural and chemical activators of Nrf2 on inflammation, oxidative stress, macrophage polarization, and bactericidal activity in an in vitro macrophage infection model[J]. PloS one, 2020,15(6):e234484.
[9] Machlus K R, Italiano J J. The incredible journey: From megakaryocyte development to platelet formation[J]. J Cell Biol, 2013,201(6):785-796.
[10] Hitchcock I S, Kaushansky K. Thrombopoietin from beginning to end[J]. British journal of haematology, 2014,165(2):259-268.
[11] Josefsson E C, Dowling M R, Lebois M, et al. The Regulation of Platelet Life Span[J]. Platelets (Third Edition), 2013,8(1):51-65.
[12] Grozovsky R, Giannini S, Falet H, et al. Regulating billions of blood platelets: glycans and beyond[J]. Blood, 2015,126(16):1877-1884.
[13] Lefran Ais E, Ortiz-Mu Oz G, Caudrillier A, et al. The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors[J]. Nature, 2017,544(7648):105-109.
[14] Raymond S L, Holden D C, Mira J C, et al. Microbial recognition and danger signals in sepsis and trauma[J]. Biochim Biophys Acta Mol Basis Dis, 2017,1863(10 Pt B):2564-2573.
[15] Peters C G, Michelson A D, Flaumenhaft R. Granule exocytosis is required for platelet spreading: differential sorting of α-granules expressing VAMP-7[J]. Blood, 2012,120(1):199-206.
[16] Thomas M R, Storey R F. Effect of P2Y12 inhibitors on inflammation and immunity[J]. Thrombosis and haemostasis, 2015,114(3):490.
[17] Tylek T, Schilling T, Schlegelmilch K, et al. Platelet lysate outperforms FCS and human serum for co-culture of primary human macrophages and hMSCs[J]. Scientific reports, 2019,9(1):3533.
[18] R?szer T, Keisari Y. Understanding the Mysterious M2 Macrophage through Activation Markers and Effector Mechanisms[J]. Mediators of inflammation, 2015,2015:1-16.
[19] Andonegui G, Kerfoot S M, McNagny K, et al. Platelets express functional Toll-like receptor-4[J]. Blood, 2005,106(7):2417-2423.
[20] Cauwenberghs N, Vanhoorelbeke K, Vauterin S, et al. Structural determinants within platelet glycoprotein Ibalpha involved in its binding to von Willebrand factor[J]. Platelets, 2000,11(7):373-378.
[21] Zarbock A, Singbartl K, Ley K. Complete reversal of acid-induced acute lung injury by blocking of platelet-neutrophil aggregation[J]. The Journal of clinical investigation, 2006,116(12):3211-3219.
[22] Narayanan P K, Li N. In Vitro Monocyte/Macrophage Phagocytosis Assay for the Prediction of Drug-Induced Thrombocytopenia[J]. Curr Protoc Toxicol, 2019,79(1):e68.
[23] Simon D I, Chen Z, Xu H, et al. Platelet glycoprotein ibalpha is a counterreceptor for the leukocyte integrin Mac-1 (CD11b/CD18)[J]. J Exp Med, 2000,192(2):193-204.
[24] Zarbock A, Polanowska-Grabowska R K, Ley K. Platelet-neutrophil-interactions: Linking hemostasis and inflammation[J]. Blood reviews, 2006,21(2):99-111.
[25] Y W, H G, C S, et al. Corrigendum: Leukocyte integrin Mac-1 regulates thrombosis via interaction with platelet GPIbα.[J]. Nature communications, 2017,8:16124.
[26] Wong C H, Jenne C N, Petri B, et al. Nucleation of platelets with blood-borne pathogens on Kupffer cells precedes other innate immunity and contributes to bacterial clearance[J]. Nat Immunol, 2013,14(8):785-792.
[27] Carestia A, Mena H A, Olexen C M, et al. Platelets Promote Macrophage Polarization toward Pro-inflammatory Phenotype and Increase Survival of Septic Mice[J]. Cell reports (Cambridge), 2019,28(4):896-908.
[28] Strauss R, Wehler M, Mehler K, et al. Thrombocytopenia in patients in the medical intensive care unit: bleeding prevalence, transfusion requirements, and outcome[J]. Crit Care Med, 2002,30(8):1765-1771.
[29] Narayanan P K, Li N. In Vitro Monocyte/Macrophage Phagocytosis Assay for the Prediction of Drug-Induced Thrombocytopenia[J]. Curr Protoc Toxicol, 2019,79(1):e68.
[30] Xiang B, Zhang G, Guo L, et al. Platelets protect from septic shock by inhibiting macrophage-dependent inflammation via the cyclooxygenase 1 signalling pathway[J]. Nat Commun, 2013,4:2657.
[31] Yan R, Chen M, Ma N, et al. Glycoprotein Ibalpha clustering induces macrophage-mediated platelet clearance in the liver[J]. Thromb Haemost, 2015,113(1):107-117.
[32] Raymond S L, Holden D C, Mira J C, et al. Microbial recognition and danger signals in sepsis and trauma[J]. Biochimica et biophysica acta. Molecular basis of disease, 2017,1863(10):2564-2573.
[33] Barrett T J, Schlegel M, Zhou F, et al. Platelet regulation of myeloid suppressor of cytokine signaling 3 accelerates atherosclerosis[J]. Science translational medicine, 2019,11(517):x481.
[34] Yagnik D, B L S, Slomiany B L. Macrophage Derived Platelet Activating Factor Implicated in the Resolution Phase of Gouty Inflammation[J]. International journal of inflammation, 2014,2014(2014):526496.
[35] Johnson C D. Platelet-Activating Factor and Platelet-Activating Factor Antagonists in Acute Pancreatitis[J]. Digestive surgery, 1999,16(2):93-101.
[36] Ersoy B, Huseyinov A, Darcan S. The role of platelet-activating factor in pathogenesis of type 1 diabetes[J]. Diabetes Care, 2005,28(4):980.
[37] Schlondorff D, Neuwirth R. Platelet-activating factor and the kidney[J]. Am J Physiol, 1986,251(1 Pt 2):F1-F11.
[38] Muehlmann L A, Michelotto P V, Nunes E A, et al. PAF increases phagocytic capacity and superoxide anion production in equine alveolar macrophages and blood neutrophils[J]. Research in veterinary science, 2012,93(1):393-397.
[39] Prescott S M, Zimmerman G A, Stafforini D M, et al. Platelet-activating factor and related lipid mediators[J]. Annu Rev Biochem, 2000,69:419-445.
[40] Ishii S, Kuwaki T, Nagase T, et al. Impaired Anaphylactic Responses with Intact Sensitivity to Endotoxin in Mice Lacking a Platelet-activating Factor Receptor[J]. The Journal of experimental medicine, 1998,187(11):1779-1788.
[41] Maier R V, Hahnel G B, Fletcher J R. Platelet-activating factor augments tumor necrosis factor and procoagulant activity[J]. J Surg Res, 1992,52(3):258-264.
[42] McManus L M, Hanahan D J, Demopoulos C A, et al. Pathobiology of the intravenous infusion of acetyl glyceryl ether phosphorylcholine (AGEPC), a synthetic platelet-activating factor (PAF), in the rabbit[J]. The Journal of immunology (1950), 1980,124(6):2919.
[43] De Plaen IG, XB H, X L, et al. Lipopolysaccharide induces CXCL2/macrophage inflammatory protein-2 gene expression in enterocytes via NF-kappaB activation: independence from endogenous TNF-alpha and platelet-activating factor.[J]. Immunology, 2006,118(2):153-163.
[44] Hwaiz R, Rahman M, Syk I, et al. Rac1-dependent secretion of platelet-derived CCL5 regulates neutrophil recruitment via activation of alveolar macrophages in septic lung injury[J]. J Leukoc Biol, 2015,97(5):975-984.
[45] Terkeltaub R, Zachariae C, Santoro D, et al. Monocyte-derived neutrophil chemotactic factor/interleukin-8 is a potential mediator of crystal-induced inflammation[J]. Arthritis and rheumatism, 1991,34(7):894-903.
[46] Hwaiz R, Rahman M, Syk I, et al. Rac1-dependent secretion of platelet-derived CCL5 regulates neutrophil recruitment via activation of alveolar macrophages in septic lung injury[J]. J Leukoc Biol, 2015,97(5):975-984.
[47] Aswad M, Assi S, Schif-Zuck S, et al. CCL5 Promotes Resolution-Phase Macrophage Reprogramming in Concert with the Atypical Chemokine Receptor D6 and Apoptotic Polymorphonuclear Cells[J]. The Journal of immunology (1950), 2017,199(4):1393-1404.
[48] Huynh M L, Fadok V A, Henson P M. Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-beta1 secretion and the resolution of inflammation[J]. J Clin Invest, 2002,109(1):41-50.
[49] Byrne A, Reen D J. Lipopolysaccharide Induces Rapid Production of IL-10 by Monocytes in the Presence of Apoptotic Neutrophils[J]. The Journal of immunology (1950), 2002,168(4):1968-1977.
[50] Mitchell S, Thomas G, Harvey K, et al. Lipoxins, aspirin-triggered epi-lipoxins, lipoxin stable analogues, and the resolution of inflammation: stimulation of macrophage phagocytosis of apoptotic neutrophils in vivo[J]. J Am Soc Nephrol, 2002,13(10):2497-2507.
[51] Chapman L M, Aggrey A A, Field D J, et al. Platelets Present Antigen in the Context of MHC Class I[J]. The Journal of immunology (1950), 2012,189(2):916-923.
[52] Hilt Z T, Pariser D N, Ture S K, et al. Platelet-derived β2M regulates monocyte inflammatory responses[J]. JCI insight, 2019,4(5).
[53] Hilt Z T, Ture S K, Mohan A, et al. Platelet-derived beta2m regulates age related monocyte/macrophage functions[J]. Aging (Albany NY), 2019,11(24):11955-11974.
[54] Kasper B, Petersen F. Molecular pathways of platelet factor 4/CXCL4 signaling[J]. European journal of cell biology, 2011,90(6):521-526.
[55] Lindsey M L, Jung M, Yabluchanskiy A, et al. Exogenous CXCL4 infusion inhibits macrophage phagocytosis by limiting CD36 signalling to enhance post-myocardial infarction cardiac dilation and mortality[J]. Cardiovasc Res, 2019,115(2):395-408.
[56] Schwartzkopff F, Grimm T A, Lankford C S R, et al. Platelet factor 4 (CXCL4) facilitates human macrophage infection with HIV-1 and potentiates virus replication[J]. Innate immunity (London, England), 2009,15(6):368-379.
[57] Pervushina O, Scheuerer B, Reiling N, et al. Platelet Factor 4/CXCL4 Induces Phagocytosis and the Generation of Reactive Oxygen Metabolites in Mononuclear Phagocytes Independently of Gi Protein Activation or Intracellular Calcium Transients[J]. The Journal of immunology (1950), 2004,173(3):2060-2067.
[58] Krauel K, Potschke C, Weber C, et al. Platelet factor 4 binds to bacteria, [corrected] inducing antibodies cross-reacting with the major antigen in heparin-induced thrombocytopenia[J]. Blood, 2011,117(4):1370-1378.
[59] Maier S, Traeger T, Entleutner M, et al. CECAL LIGATION AND PUNCTURE VERSUS COLON ASCENDENS STENT PERITONITIS: TWO DISTINCT ANIMAL MODELS FOR POLYMICROBIAL SEPSIS[J]. Shock, 2004,21(6):505-512.
[60] Kelton, John G. Heparin-induced thrombocytopenia[J]. Postgraduate Medicine, 1986,16(2):173-186.
[61] Nguyen T H, Medvedev N, Delcea M, et al. Anti-platelet factor 4/polyanion antibodies mediate a new mechanism of autoimmunity[J]. Nat Commun, 2017,8:14945.
[62] Pongas G, Dasgupta S K, Thiagarajan P. Antiplatelet factor 4/heparin antibodies in patients with gram negative bacteremia[J]. Thrombosis Research, 2013,132(2):217-220.
[63] Gleissner C A. Macrophage Phenotype Modulation by CXCL4 in Atherosclerosis[J]. Frontiers in physiology, 2012,3:1.