Neutrophils contribute to IgG-dependent anaphylaxis in FcγR-humanised mice 

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Myeloid cells of the innate immune system

Myeloid cells are, in the most part, derived from precursors in the bone marrow. A common myeloid progenitor cell gives rise to granulocytes, including neutrophils, monocytes, erythrocytes and platelets. Recent data suggests an early developmental separation of a neutrophil and monocyte precursor (NMP), at the same time as the common lymphoid precursor (CLP), from an alternate lineage giving rise to megakarayocytes and erythrocytes (MEP) and eosinophils, mast cells and basophils (EMP) [2, 3]. At different stages of development myeloid cells can migrate to take up residence in the tissues; resident tissue cell populations coexist with those recruited during inflammation or injury.
Considering the function of myeloid cells in immunity, it is apparent that the diverse and specialised roles of phagocytes and granulocytes create an intricate network of host defence. Significant phenotypic plasticity is afforded between homeostatic and inflammatory conditions, as well as between tissue resident cells and those that are recruited during an inflammatory response [4]. Nearly every cell type has its canonical function, or that for which it was originally identified; yet most do far more than that. This introduction will address some of the most pertinent aspects of the innate myeloid system to provide a context for the studies of this thesis work. Firstly, neutrophils as a major focus, secondly monocyte/macrophages, and thirdly other granulocyte populations mast cells, basophils and eosinophils. Each of these populations has been suggested to contribute to local and systemic pathological inflammation.
It is particularly relevant to appreciate how myeloid cells can detect and respond to inflammatory stimuli, whether derived from pathogen associated molecular patterns (PAMPs) or host-derived danger associated molecular patterns (DAMPs). PAMP recognition is achieved primarily by pattern recognition receptors (PRRs), which may be expressed on the cell surface or as soluble opsonising factors: for example Toll-like receptor 4 (TLR4) that recognises the lipopolysaccharide (LPS) of gram-negative bacteria. Antibodies represent a crucial link between adaptive and innate immunity: they confer the innate myeloid cell system with an adaptive specificity, and myeloid cells express numerous cell surface antibody receptors.

Neutrophils

Fifty to seventy percent of circulating leukocytes in human blood are neutrophils. In mice housed in Specific Pathogen Free (SPF) conditions this figure is closer to ten to twenty-five percent, yet neutrophil numbers can increase dramatically during infection or following an inflammatory stimulus. Mature neutrophils have a diameter of 7–10μm and possess a segmented nucleus, leading to their alternate alias PolyMorphoNuclear cells (PMNs). As granulocytes, their cytoplasm is enriched with granules and secretory vesicles. Neutrophils are defined in human blood by CD66 and CD15 expression. In the mouse, neutrophils are distinguished by high levels of expression of CD11b, a component of Mac-1 integrin expressed by the majority of myeloid cells, and the surface marker Granulocyte antigen 1 (Gr-1), which comprises two molecules Ly-6C and Ly-6G. Neutrophils express intermediate levels of Ly-6C, which can also be prominently expressed by monocytes, but distinctively high levels of Ly-6G, and therefore the latter is considered a unique surface marker.
In order to examine the role of neutrophils in inflammation, several important aspects of these cells will be introduced in the following sections: firstly, neutrophil development and granule formation and release; then, their lifespan in circulation, release from the bone marrow and turnover; and their recruitment and migration. Furthermore, the mechanisms of microbial killing by neutrophils will be described, as well as the perceived phenotypic heterogeneity of these cells; the former which is associated with inflammatory cues or tissue damaging effects, and the latter which may determine neutrophil involvement in an ongoing inflammatory response.

Neutrophil development and granulopoiesis

Mature neutrophils differentiate from hematopoietic precursors (HSCs) in the bone marrow. The daily production of neutrophils is up to 1011 in healthy individuals, a figure that can increase several-fold during infection [5]. Granulocyte colony stimulating factor (G-CSF) supports neutrophil production and is essential for increasing this production during infection; yet is not absolutely required for neutrophil development as G-CSF knock-out mice still generate mature neutrophils at about 25% of normal levels. A transcriptional regulatory network governed mostly by the transcription factors PU.1 and C/EBP controls the differentiation of HSCs towards granulocytes. PU.1 is necessary for myeloid commitment, and thereafter the balance between PU.1 and C/EBP controls lineage commitment [6, 7]. The transcription factor growth factor independent-1 (Gfi-1) is necessary for neutrophil differentiation [8, 9]. Upregulated during commitment to the granulocyte lineage, Gfi-1 represses the monocyte-promoting transcription factor Egr2 [10] and the gene Csf1, coding for the cytokine CSF-1 which supports monocyte development [11] (Figure 1.1). Mutations in Gfi1 that affect its transcriptional repressor activity lead to severe neutropenia, in humans [12] and mice [9, 13], a phenotype which has been applied to study the effect of neutrophil absence in different mouse models. After 4-6 days in the bone marrow, neutrophils are released into the circulation from the post-mitotic pool (Figure 1.1) (see section Neutrophil lifespan and aging).
Neutrophils are filled with granules and secretory vesicles, which play a pivotal role in neutrophil function [14, 15]. Granules are stores of proteins containing membrane surface receptors, antimicrobial products, and enzymes to degrade the extracellular matrix, encapsulated within a phospholipid bilayer membrane and an intragranular membrane. Their contents may be destined for exocytosis or fusion with the phagosome [14, 15]. The large majority of neutrophil functions, from migration to extravasation to microbial killing, are guided by the mobilization of cytoplasmic granules and secretory vesicles [5].
The developing neutrophil passes through several stages: myeloblast, promyelocyte, myelocyte, metamyelocyte, band cell and, finally, polymorphonuclear (segmented) cell (Figure 1.1). Granule biogenesis is intrinsically linked with neutrophil development: the heterogeneity of granule subsets can be described based on their protein content as well as their sequential formation. According to the ‘targeted by timing’ model of granulopoiesis, different granule proteins are synthesised during different stages of development, resulting in discrete granule subsets with different composition, of which at least three major subsets have been defined [16-20] (Figure 1.1).
Primary granules, appearing first, have a high content of myeloperoxidase (MPO) and have been designated alternatively “peroxidase positive” or “azurophil” granules. As well as MPO, primary granules contain alpha-defensins, bacteriocidal proteins, and serine proteases [14]. The three neutrophil serine proteases, proteinase-3, cathepsin G, and elastase are structurally similar, exhibit proteolytic activity against components of the extracellular matrix, and induce the activation of other leukocytes, endothelial and epithelial cells, and platelets [21]. Primary granules appear to undergo limited exocytosis and predominantly mediate microbe killing in the phagolysomes.
As MPO synthesis stops at the promyelocyte/myelocyte transition, granules that form later are peroxidase negative. Secondary, or specific, granules contain high levels of lactoferrin and collagenase and form at the myelocyte or metamyelocyte stage, whereas tertiary granules have a high gelatinase content and form in band cells and hypersegmented neutrophils (Figure 1.1).
Figure 1.1: Neutrophil development and granulopoiesis. Neutrophils and monocytes develop from a common myeloid precursor; the transcriptional repressor Gf1 inhibits Egr2 and Csf1 to promote development of the neutrophil lineage (upper panel). From the myeloblast to the mature neutrophil stage, granules are formed chronologically, and biosynthetic windows of granule protein synthesis determine the composition of primary (red) secondary (blue) and tertiary (yellow) granules (lower panel). Secretory vesicles (green) are formed only during the last stages of neutrophil maturation, and contain numerous cell surface receptors synthesised at that time, including CD14, CD16 and CD11b. MPO, myeloperoxidase; PR3, proteinase 3; NE, neutrophil elastase. Adapted from [5, 14] using the online resource Servier Medical Art.
Moreover, secondary granules are larger and full of antibiotic agents such as lactoferrin and lysozyme. Neutrophil matrix mellaproteases (MMPs) include collagenase (MMP8) of secondary granules [22], gelatinase (MMP9) of tertiary granules, and leukolysion (MT6-MMP/MMP- 25), which is distributed across these two subsets and is also contained in secretory vesicles [23]. These MMPs enable degradation of the vascular basement membrane and interstitial structures to facilitate neutrophil recruitment and migration. The exocytosis of granules is a tightly regulated hierarchical process [15], which occurs inversely to their formation: as such, tertiary granules are more easily exocytosed than secondary granules. This aligns with a general model whereby tertiary granules are a reservoir of enzymes (gelatinase) and surface receptors necessary for neutrophil extravasation and migration, while secondary granules are mobilised for migration within the tissue (collagenase) and antimicrobial functions, whether by fusion to the phagosome or to the exterior of the cell [14, 21, 24]. Thus, although neutrophils are armed with an arsenal of cytotoxic and potentially tissue-damaging agents, in an inflammatory response the regulated exocytosis of granules permits the targeted release of their contents, minimizing collateral damage.
Secretory vesicles, on the contrary to granules, are formed by endocytosis during the late stages of neutrophil maturation, and are rapidly released upon chemotactic stimulation [25, 26]. Upon release, their membrane is incorporated with the surface membrane of the neutrophil; thereby, secretory vesicles are seen to be critical reservoirs of membrane surface receptors necessary for the early stages of neutrophil activation and adherence to the vasculature. These include β2-integrin CD11b/ CD18 (Mac-1, CR3) [27], the complement receptor 1 (CR1)[28], the receptor for endotoxin CD14 and the IgG receptor FcRγIII (CD16) [29]. In this manner, secretory vesicles can transform a resting neutrophil, with few surface receptors and minimal responsiveness to soluble mediators, to an extremely responsive cell [30].

Lifespan, margination and aging

Always considered as short lived cells, with a half-life of just several hours in the blood, 1.5h for mice and 8h for humans, recent evidence suggests that neutrophils can in fact live much longer in the circulation, with a lifespan up to 12.5h for mice and 5.4 days for humans; although the latter findings have been subjected to criticism [31]. Certainly the real figure lies somewhere in between, yet the lifespan of neutrophils is extended after activation, by inhibition of apoptosis pathways [32]. Some data also suggests that mature neutrophils have a limited proliferation potential after exiting the bone marrow; but, most importantly, a prolonged neutrophil lifespan endows them with the capacity to engage in more complex activities within the tissue [33].
Neutrophil release from the bone marrow is under differential control during steady state or inflammation. The retention of neutrophils in the bone marrow is controlled by the CXCL12/CXCR4 chemokine axis and VLA-4/VCAM-1 interactions. G-CSF stimulates neutrophil release from the bone marrow, in part by inhibiting macrophage expression of VCAM-1 and CXCL12. One paradigm of maintaining leukocyte homeostasis considers that phagocytosis of dying neutrophils in the periphery provides negative feedback for their mobilisation from the bone marrow via an IL-23/IL-17/G-CSF cytokine axis [34]. A model of antagonistic signalling via CXCR4 and CXCR2 accounts for the rapid mobilisation of neutrophils during inflammation and infection [35, 36]: ligands for CXCR2, CXCL1 and CXCL2, expressed by bone marrow endothelial cells promote neutrophil retention in the steady state; whereas inflammatory mediators and chemokines in the periphery promote dominant CXCR2 signalling and release, and G-CSF signalling further favours neutrophil release [5, 37]. Dendritic cells can regulate neutrophil distribution between the bone marrow and peripheral organs, regulating the production of G-CSF, CXCL1 and CCL2 [38].
An alternative paradigm removes the distinction between ‘steady state’ and ‘emergency’ granulopoiesis, and postulates rather that signalling via pattern recognition molecules modulates both [39]. Healthy mice in normal conditions have more circulating neutrophils than those raised in aseptic housing; and neutropenia induced by antibody-mediated depletion raises G-CSF and stimulates granulopoiesis, but not in TLR4-deficient animals [40]. This model aligns with recent data indicating that the microbiome also regulates neutrophil aging [41].
Neutrophils can be found abundantly in the lung, spleen, liver and bone marrow under physiological conditions. The concentration of neutrophils in peripheral organs is referred to as ‘organ-marginated’ pools, and these may represent important neutrophil reservoirs for rapid deployment, in addition to the bone marrow. In the case of the lung-marginated pool, neutrophils persist in the pulmonary vasculature much longer than accounted for by mean intravascular transit time, and it appears that these cells are actively patrolling the tissue, rather than merely trapped in the microvasculature [33]. Certainly neutrophils can be rapidly mobilised from the lung: adherence to the pulmonary vasculature is mediated by CXCR4-CXCL12 interactions, and the numbers of neutrophils in circulation can be boosted by CXCR4 blockade, which both mobilises neutrophils from the lung and blocks their return to the bone marrow [42].
CXCR4 is expressed at low levels on circulating neutrophils, and its upregulation is posited to promote return to the bone marrow and clearance therein. In mice, a senescent or ‘aged’ neutrophil population is characterised by the up regulation of surface CXCR4 and CD11b, decrease in CD62L,
reduced size and nucleus hypersegmentation [43]. The homeostatic turnover of neutrophils fluctuates throughout the day, and controls circadian oscillations in hematopoietic stem cell mobilisation [43]. The increase in CXCR4 on aged neutrophils would seem to suggest that these cells preferentially populate the pulmonary marginal pool. Neutrophil aging is regulated by the microbiota, and signalling via TLRs; as such, aging predisposes to neutrophil overactivation, indicated by high CD11b expression and a greater propensity for the formation of neutrophil extracellular traps (NETs) [41]. Altogether, it is important to bear in mind that models of constitutive or inducible neutrophil depletion, such as will be used herein, likely have systemic effects on cytokine release and the hematopoietic niche. Furthermore, that depending on the age of the neutrophil these cells may exhibit different reactivity in vivo.

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Neutrophil recruitment into the tissue

The neutrophil inflammatory recruitment cascade and the mechanisms underlying neutrophil extravasation from the bloodstream into the tissues have been well elucidated, and classically involve the steps of tethering, rolling, adhesion, crawling, and finally transmigration [33]. Changes to the vascular endothelium comprising the upregulation of P-selectin and L-selectin occurs following the release of inflammatory mediators by local leukocytes, including histamine, cysteinyl-leukotrines and cytokines, or by direct endothelial cell PRR engagement. P-selectin and L-selectin on endothelial cells bind to their ligands on neutrophils, including P-selectin glycoprotein ligand 1 (PSGL-1), to capture neutrophils from the blood flow. This manner of tethering leads to neutrophils rolling along the endothelium, under shear stress, and associated expression of lymphocyte function-associated antigen 1 (LFA1), which binds to intercellular adhesion molecule 1 (ICAM1) and ICAM2 on the endothelium to promote cell arrest.
Firm adhesion of tethered neutrophils to the endothelium occurs following neutrophil priming by inflammatory cytokines, chemoattractants, or PAMPs. Neutrophils express the integrins LFA1 ( 1 2; that is 2 integrin CD11a complexed with CD18) and Mac1 ( M 2; 2 integrin CD11b with CD18), which bind endothelial surface molecules ICAM1 and ICAM2. Chemokine receptor signalling on neutrophils induces changes in the conformation of these integrins, a phenomenon referred to as inside-out signalling, to increase their affinity for their respective ligands, as well as an increase in surface CD11b from intracellular stores. Adherent neutrophils initiate probing behaviour, and crawl along the endothelium to reach an appropriate site for extravasation, likely guided by chemokines immobilised on the endothelium.
Extravasation of neutrophils involves the crossing of the endothelium, and then the basement membrane, a process referred to as transmigration and dependent on integrins, ICAM1 and ICAM2, and vascular cell adhesion protein 1 (VCAM1) as well as interactions with a variety of junctional proteins. Endothelial cells may also undergo cytoskeletal changes to facilitate neutrophil transmigration. One common model is that neutrophils cross the basement membrane via the release of granules containing active proteases – yet evidence for a protease requirement in this process is in fact scarce [5, 33]. Rather, neutrophils may emigrate through more porous and less dense regions of the basement membrane [44].
Unique strategies for neutrophil extravasation evidently correspond to different specific vascular beds. In the pulmonary circulation, low blood velocity means that neutrophil extravasation can occur in the small capillaries. The liver sinusoidal endothelium is particularly porous, and the narrow vasculature also results in a large marginated pool of neutrophils. Recruitment via the portal venules is distinct to the sinusoidal capillaries. Finally, described above are the strategies for transendothelial neutrophil migration, much of which has been observed by in vivo imaging, however transepithelial migration also occurs and is particularly relevant at mucosal surfaces, for example the lung and the gut. Transepithelial migration requires the leukocyte integrins Mac1 and LFA1, but not the apically expressed ICAM-1/VCAM-1 [45]. The triggering receptor expressed on myeloid cells (TREM)-1 was implicated in transepithelial migration associated with acute lung injury [46]. Together these considerations imply that neutrophils in the tissue, and indeed different tissues, having undergone the sequential steps of extravasation and migration, have a different phenotypic and activation profile compared to naïve cells in the circulation.

Multidirectional migration

Neutrophil reverse transmigration, that is the capacity for neutrophils to migrate from the tissue back to the bloodstream, was originally demonstrated in vitro: after ablumunial to luminal migration, neutrophils exhibited an ICAM-1hi phenotype, prolonged lifespan, and increased capacity for superoxide production, and phenotypically similar neutrophils could be found in the circulation 47]. Thereafter, in vivo neutrophil reverse transmigration was described in the context of ischemia-reperfusion injury or high levels of leukotriene B4 (LTB4) [48, 49]. The capacity for neutrophils to migrate out of the tissue and rejoin the circulation suggests a capacity to transmit inflammatory signals and systemically influence responses. Indeed, ICAM-1hi neutrophils were identified in organs distal to the site of inflammation [48, 49].
Neutrophils can also exit the tissue via the lymphatics, and thereby shuttle antigen from sites of exposure, such as the skin, to secondary lymphoid organs following infection or immunisation [50-52]. Lymph node migration by neutrophils is in accordance with their capacity to promote some T cell responses, as well as to regulate antigen presentation and the extent of lymphocyte proliferation [51-55].

Killing mechanisms of neutrophils: phagocytosis, oxidative burst, MPO & NETs

Phagocytosis is the hallmark of innate immune defence, involving the engulfment of living pathogens or other targets, most notably by macrophages, but also by neutrophils. Pathogen recognition via direct PRRs or ligation of receptors for humoral factors (such as receptors for IgG, FcγR) results in actin polymerization and cytoskeletal remodelling such that the phagocyte can extend its plasma membrane. The resulting pseudopodia structure surrounds the target and fuses to encapsulate the target within an intracellular vesicle called a phagosome. In macrophages, phagosomal acidification and fusion with lysosomes, oxidative compartments filled with antimicrobial enzymes and proteins, leads to the killing, degradation and elimination of the engulfed target [56]. In neutrophils, the phagosomal pH remains alkaline or neutral, facilitating the enzymatic action of neutrophil serine proteases. The coordinated fusion of neutrophil granules with phagosomes permits the targeting of potent and toxic granule proteins to the ingested target, minimizing release into the extracellular milieu [15]. Conversely, engagement of neutrophil phagocytic receptors by IgG or complement deposited on large surfaces, which cannot be engulfed, results in ‘frustrated phagocytosis’, and the extracellular release of granule contents and oxidative products: a highly inflammatory and potentially damaging outcome [45].
The generation of reactive oxidants via the NADPH oxidase system is vital for optimal neutrophil microbicidal activity [39]. Its assembly and activity in phagocytes requires the translocation of a cytoplasmic complex containing p47phox, p67phox and p40phox to the membrane bound heterodimer of gp91phox and p22phox [57, 58]. In a resting state, the gp91phox -p22phox heterodimer (the flavocytochrome b558) resides predominantly in the membrane of neutrophil secondary (specific) granules (Figure 1.1). Regulation of the NADPH oxidase is thereby achieved by spatial segregation of its components, and no activity is detected in resting cells. Activation of the phagocyte triggers phosphorylation of p47phox to permit full assembly of the entire NADPH oxidase. Sustained NADPH oxidase activity requires a continual translocation of the cytosolic components to the membrane, and in neutrophils continual furnishing by membrane components derived from granule fusion. The NADPH oxidase generates reactive superoxide anion (O2. –) as an immediate product, and hydrogen peroxidase (H2O2) by dismutation of the superoxide. The rapid agonist- and activation-dependent assembly of the NADPH oxidase to generate reactive oxygen species (ROS) is referred to as the ‘respiratory burst’. The NADPH oxidase can also assemble at the plasma membrane, and thereby generate ROS in the surrounding tissue environment. The serious requirement for the NADPH oxidase in host defence is exemplified by patients with chronic granulomatous disease (CGD), in which mutations in components of the oxidase complex result in its impaired or absent function in phagocytes. CGD patients are predisposed to chronic and recurrent bacterial and fungal infections.
Myeloperoxidase (MPO), the major component of neutrophil primary granules, is a crucial additional weapon in the neutrophil antimicrobial arsenal. MPO catalyses the conversion of hydrogen peroxide generated by the NADPH oxidase into hypochlorous acid (HOCl), which has much greater antimicrobial potency. H2O2 has a greater redox potential than HOCl, but HOCl can kill bacteria such as E.Coli ~1000 times faster, presumably due to its extremely rapid reaction kinetics [59]. The MPO-H2O2-Cl system requires a source of chloride, provided by various phagosome-associated transporters, primarily the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is associated with neutrophil secretory vesicles, but not the plasma membrane, and is recruited by vesicle fusion during phagocytosis: another layer of regulation to direct the cytotoxic effects of neutrophils to targets within. MPO deficient neutrophils are capable of killing bacteria, and MPO deficient patients do not exhibit the severe phenotype of CGD patients. Yet the killing by MPO-deficient neutrophils is slower, depending on the pathogen, and although in many cases the pathogen is eventually eliminated, MPO-deficient animals do exhibit an increased susceptibility to bacterial and fungal infections; findings that indicate an impact of pathogen load. The lack of a strong patient phenotype is more suggestive of compensatory mechanisms. Indeed, the MPO-H2O2-Cl system has downstream effects on the collective antimicrobial activity of other neutrophil granule proteins: notably, serine proteases are inactivated by oxidation. Moreover, since MPO catalyses the consumption of H2O2, in its absence the concentration of H2O2 is increased [59].

Table of contents :

1 Introduction 
1.1 Myeloid cells of the innate immune system
1.1.1 Neutrophils
1.1.2 Monocytes, Macrophages and Mononuclear Phagocytes
1.1.3 Mast cells, Basophils and Eosinophils
1.2 Neutrophil death & inflammation resolution: a focus on lipid mediators
1.3 Antibodies & their receptors: conferring innate immune cells with adaptive specificity
1.3.1 B cells and the BCR
1.3.2 Antibodies and their classes
1.3.3 IgG antibody receptors (FcgR)
1.4 Systemic Inflammation
1.4.1 Inflammation-associated circulatory shock
1.4.2 Endotoxemia
1.4.3 Anaphylaxis
2 Summary and objectives 
3 A novel model of inducible neutropenia reveals a protective role for neutrophils during systemic inflammation 
3.1 PAPER I
4 Neutrophils contribute to IgG-dependent anaphylaxis in FcγR-humanised mice 
4.1 PAPER II
4.2 Considering high affinity FcγRI: the Audrey mouse
4.2.1 Audrey mice exhibit hFcγRI expression patterns comparable to that of humans, and retain hFcγRIIA/IIB/III expression of VG1543 mice
4.2.2 hFcγRI does not contribute to IVIG-PSA in Audrey mice, which proceeds via a dominant pathway involving hFcγRIIA, Neutrophils and PAF
4.2.3 Supplemental Methodology and Data
5 Discussion 
5.1 Part I: A protective role for neutrophils in LPS endotoxemia
5.2 Part II: The inflammatory effects of neutrophils: novel mouse models to study neutrophil function in vivo
5.2.1 A novel model of inducible neutropenia: PMNDTR mice
5.2.2 PMNDTR mice to study neutrophils in antibody-dependent pathologies: deciphering the contribution of neutrophils to systemic anaphylaxis
5.2.3 Audrey & humanised mouse models to study FcγR: limitations and potential
5.3 Part III: Neutrophils as protective or pathological agents of systemic inflammation
5.4 Part IV: Towards the clinic: systemic anaphylaxis to neuromuscular blocking drugs
5.4.1 Developing a mouse model of systemic anaphylaxis to Rocuronium Bromide
5.4.2 Evidence from the clinical study NASA: Neutrophil activation in systemic anaphylaxis
5.5 Final Considerations and Perspectives
6 References 
7 Annex 
7.1 IgG subclasses determine pathways of anaphylaxis in mice
7.2 In vivo effector functions of high-affinity mouse IgG receptor FcgRI in disease and therapy models
7.3 Review – Contribution of human FcγRs to disease with evidence from human polymorphisms and transgenic animal studies
7.4 Book chapter – Anaphylaxis (Immediate hypersensitivity): from old to new mechanisms

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