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Putting priming into perspective – from cellular heterogeneity to cellular plasticity
VIEWPOINT I M M U N O L O G Y T O D AY
Putting priming into perspective – from cellular heterogeneity to cellular plasticity Claus Kroegel, Martin Foerster, Daniela Häfner, P. Reinhard Grahmann, Jane A. Warner and Ruedi Braun The concept of priming is widely used in cell biology and has come to
U
nder certain in vivo and mediators, cell viability, adhesion, and mean the functional enhancement of in vitro conditions, the funcchemotaxis8,9. Similar spectra of cellular a given cell by cytokines. ÔPrimedÕ function enhanced by priming have also tion of inflammatory cells cells have a number of other cellular been observed for macrophages, neutrophils can be enhanced. This has and basophils. attracted much attention, as it explains the alterations, although the discrepancy between the high agonist conrelationship between functional and centrations required to elicit a cell response phenotypical diversity has not been Priming is achieved by soluble mediators in vitro and the relatively low concentrations Originally, priming was associated with the achieved in vivo. established. Here, Claus Kroegel and action of cytokines, and several in vitro studThe ability of cells to adopt an increased colleagues discuss the dynamic ies have shown that culturing eosinophils in functional status under certain conditions or the presence of interleukin 3 (IL-3), IL-5, in a defined environment is referred to as nature of inflammatory-cell granulocyteÐmacrophage colony-stimulating ÔprimingÕ. In addition to the functional varipriming, which might be part of a factor (GM-CSF), tumour necrosis factor a ability, priming has been closely associated broader means of comprehending (TNF-a) or interferon g makes the cell funcwith morphological, physical and phenotionally hyperactive8Ð10. However, this concept typical changes of the cell, providing the cell function in disease. needs to be modified in several ways. basis for the concept of cellular heterogeneity. First, in addition to their priming action, Although inflammatory-cell heterogeneity might be due largely to the site-specific blending of factors generated cytokines have also been shown to activate cellular effector functions within the local microenvironment, the relationship between prim- fully, including degranulation and other cellular responses11. Second, ing and other cellular alterations is still uncertain at present. There- mediators and agents that were known to activate eosinophils, such fore, this article brings together the various aspects of priming and as platelet-activating factor (PAF), complement factor 5a (C5a) and the chemotactic peptide N-formylmethionylphenylalanine (fMLP), discusses their relationship with other features of cellular diversity. have also been shown to prime eosinophils when applied at suboptimal (picomolar) concentrations12. Third, cell-permeable analogues What exactly is priming? of second messengers, phorbol ester, the calcium ionophore A23187, Priming is a feature of different cell types zymosan-activated serum and bacterial lipopolysaccharides have all The original concept of priming dates back to the observation by been shown to prime leukocytes13. Johnston et al.1 that macrophages show an enhanced oxidative burst The above findings indicate that the effect of a given soluble when exposed to proteases in vitro. However, priming is not re- agent (e.g. priming or activating) is determined by its actual concenstricted to macrophages but has also been observed in neutrophils2, tration rather than by its chemical nature, although other factors basophils3, lymphocytes4 and eosinophils5. Thus, priming represents such as the presence of cosecreted mediators, adhesive interactions a general phenomenon and is a functional option for many, if not all, with neighbouring cells or with intercellular matrix proteins might cell types. As eosinophils appear to respond to priming agents in a also play a role14,15. Therefore, the original concept that soluble very pronounced way, much of the information about priming was agents can be exclusively divided into priming and activating agents is not justified and should be abandoned. obtained with this cell type5Ð8.
Various cellular effector mechanisms are sensitive to priming
Priming by engagement of adhesion molecules
Most experiments dealing with priming assess only one or, occasionally, two cellular responses. However, priming is not restricted to a single cell function but covers myriad responses that a given cell is capable of performing. In the case of eosinophils, cellular properties induced by priming include respiratory burst, toxicity to host tissue and invading parasites, degranulation, formation of lipid
Eosinophils from allergic donors generate significantly more superoxide radicals in response to fMLP stimulation after adhering to a surface coated with vascular cell adhesion molecule 1 (Ref. 16). Monoclonal anti-CD18 antibodies prevented this adhesion-dependent priming effect, suggesting that this b2-integrin subunit is involved. Moreover, activation of the respiratory burst in human blood
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neutrophils exposed to fMLP, TNF-a or GM-CSF could only be achieved by crosslinking CD18 on the surface of the cells16. Another recent finding suggests that engagement of the L-selectin adhesion receptor serves as a priming signal, promoting subsequent firm cellto-cell adhesion17. These results clearly show that certain adhesion molecules are capable of priming inflammatory effector cells, although their mode of action is not fully understood.
How does priming relate to function and to other cellular alterations? Priming is associated with morphological alterations Eosinophils from patients with eosinophilia are morphologically distinct from cells from healthy subjects7Ð10. The changes include the appearance of cytoplasmic vacuoles, alterations in cell size and shape, an increase in granule size, solubilization and loss of granular-core and matrix proteins, and an increase in smooth tubules and vesicles18. Intracellular morphological alterations observed in vivo can also be induced by exposing eosinophils to cytokines, C5a or PAF in vitro19Ð21, suggesting that morphological heterogeneity is a general feature of inflammatory cells and is caused by inflammatory mediators.
Priming is related to physical cell changes Blood from patients with eosinophilia-associated diseases contains a population of eosinophils that segregates at a lower buoyant density when centrifuged through Percoll or Metrizamide gradients; these are known as ÔhypodenseÕ, Ôlight-densityÕ or Ôlow-densityÕ eosinophils7,8. In addition, eosinophils recovered from both pleural and bronchoalveolar spaces contain a high proportion of hypodense cells. Hypodense eosinophils are larger than normal ones, contain fewer partially lucent granules and have increased levels of both adhesion molecules and function5, revealing a remarkable resemblance to in-vitro-induced hypodense eosinophils9. Furthermore, the hypodense subpopulation often shows a variably enhanced functional response, either spontaneously or following stimulation in vitro. Moreover, normodense blood eosinophils (i.e. those with normal density) shift towards the hypodense subtype when cultured in the presence of IL-3, IL-5, GM-CSF, C5a, PAF or fMLP at both activating and priming concentrations5,9,19.
Priming is accompanied by phenotypic changes Inflammatory cells have the inherent ability to modify their expression of surface receptors. In eosinophils, for instance, a number of membrane antigens (including the complement receptors and adhesion molecules) are constitutively expressed and might be upregulated on exposure to mediators22. By contrast, other constitutively expressed surface antigens (such as L-selectin) are shed during eosinophil activation and transendothelial migration23. Finally, certain surface antigens are newly synthesized and expressed in primed or activated eosinophils, including class II proteins of the major histocompatibility complex, CD4, intercellular adhesion molecule 1
(ICAM-1) and IL-2 receptor (IL-2R)8,22Ð24. Again, these changes typically occur in vitro after the cells are cultured with cytokines known to prime eosinophils, such as IL-3, IL-5, IL-13 and GM-CSF7,20Ð23. A similarly altered phenotype can be observed in eosinophils recovered from bronchoalveolar lavage (BAL) or the sputum or blood of patients with chronic inflammatory diseases22,25Ð27. For instance, BAL and sputum eosinophils obtained from patients with atopic asthma and eosinophilic pneumonia express ICAM-1, but L-selectin expression is lower than on blood cells. Furthermore, in both diseases, the BAL and sputum eosinophils show an upregulation of the b2 integrins CD11b and CD11c, as well as the leukocyte function-associated antigen 3 (LFA-3; CD58). Taken together, these data indicate that characteristic phenotypic changes are associated with the functional changes.
Hypodense cells that are activated are not always primed Hypodense cells obtained from asthmatics produced fewer reactiveoxygen metabolites in response to zymosan than normodense cells, as assessed by nitroblue tetrazolium reduction and chemoluminescence27,28. In addition, hypodense blood eosinophils from asthmatics produced only half the amount of leukotriene C4 (LTC4) that normal cells did29. Further, BAL eosinophils from asthmatic subjects extracted 18 h after an allergen challenge had a reduced capacity to generate both prostanoids and leukotrienes (e.g. prostaglandin D2, thromboxane A2 and LTC4) than blood eosinophils when stimulated with PAF (Ref. 25). Similarily, peripheral-blood eosinophils from asthmatics synthesized less LTC4 than cells isolated from normal subjects30. In a study that compared blood and BAL eosinophils obtained from asthmatics 48 h after allergen provocation, airway eosinophils generated significantly more superoxide on activation with fMLP than cells from normal subjects but failed to do so when activated with PAF, phorbol 12-myristate 13-acetate or the Ca21 ionophore A23187 (Ref. 26). These data suggest that hypodense airway eosinophils expressing certain surface antigens associated with priming or activation, in addition to having enhanced functional properties, reveal an unaltered or a reduced functional capacity that is stimulus dependent.
Is priming a singular event? Priming is a reversible cellular process There is mounting evidence suggesting that the enhanced functional status of a primed cell spontaneously returns to the prepriming levels (depriming). For instance, human basophils stimulated with C5a only induced the production of IL-4, IL-13 and LTC4 after pretreatment with IL-3, IL-5 or GM-CSF. In addition, the priming of eosinophils with GM-CSF reached a maximum 60 min after the agonist was added and declined thereafter31. A similar pattern has been observed with several other cells32, although the time course can depend on the priming agent employed. Whereas the priming effect of IL-3 persisted for more than 18 h (Ref. 33), the action of both IL-5 and GM-CSF disappeared within a few hours3,33. These observations suggest that priming is a reversible process and that the biological effect of priming depends on the time lag between exposure to the priming agent and the activating stimulus. The data further stress
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Fig. 1. Simplified schematic representation of post-mitotic eosinophil differentiation. After differentiation, maturation and release from the bone marrow, the cell has normal density (is ÔnormodenseÕ) and is functionally passive (1). In the absence of disease, the cell will unselectively migrate into any tissue and will eventually undergo apoptosis without having performed its effector functions (ÔageingÕ) (2). However, during disease (e.g. hypereosinophilic syndrome, parasitic infection or allergy), systemically available cytokines will initiate cellular priming (3). Depending on the type, course and severity of the underlying disorder, several options are available to the circulating cell. When appropriate stimuli are absent and priming agents are lost, the priming phase can be followed by depriming, leading to an unreactive, passive cell that will eventually die (4). When primed circulating cells passing through an area of ongoing inflammation, local mediators can trigger immediate transendothelial migration. In the continued presence of priming stimuli (adhesion molecules, matrix proteins) the migrating cell retains its functionally active state but, at the same time, adopts a hypodense phenotype and undergoes further morphological changes (5). As the cell approaches the inflammatory focus, increasing mediator concentrations induce full cellular activation and the release of mediators and other effector molecules. Complete cellular activation is followed by transient desensitization, leaving the hypodense cell in a functionally unresponsive state (6). However, if local cytokines and other mediators continue to be present within an inflammatory microenvironment, hyporeactive desensitized cells can be reprimed, regaining partial or complete effector functions (7). Finally, if no additional mediators are offered, the cells remain within the circulation, slowly adopting a hypodense phenotype (8) and transmigrating non-selectively into any tissue. Owing to the raised functional reactivity of the cell, effector molecules are released after interaction with extracellular-matrix proteins and other receptors. Abbreviations: C5a, complement factor 5a; GM-CSF, granulocyteÐmacrophage colony-stimulating factor; IL-3, interleukin 3; LTB4, leukotriene B4; PAF, platelet-activating factor. Modified from Ref. 35.
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the importance of the timing and sequence of biologically active molecules for inflammatory cell performance.
Cells are susceptible to repeated priming Although priming is generally seen as a one-off, irreversible process, there is evidence that this may not always be true. It is well known that fMLP stimulates superoxide-anion release by neutrophils, leading to cellular shape changes, and inducing CD11b-dependent cell binding to albumin-coated surfaces following priming with concentrations of PAF below the activation threshold. This priming effect spontaneously declines when fMLP is added 10 min after PAF and disappears altogether after 120 min34. Moreover, a PAF-receptor antagonist added 10 min into the PAF priming period accelerated depriming of the neutrophils. More importantly, cells that had spontaneously deprimed after PAF exposure retained their capacity to be fully reprimed by subsequent addition of PAF. These observations imply that a cell can undergo priming repeatedly.
Priming can restore the function of hyporeactive cells Hypodense BAL eosinophils obtained during the late asthmatic response (18 h after endobronchial allergen challenge) synthesized less lipid mediators, either spontaneously or following stimulation, than blood cells from the same patient25. However, when the cells were pretreated with non-activating concentrations of GM-CSF for 60 min in vitro before stimulation with PAF or the Ca21 ionophore A23187, the cells regained a hyper-responsive phenotype35 (as assessed by thromboxane synthesis and LTC4 release). At the same time, their density decreased further and the expression of PAF receptors increased by more than 50%. These data suggest that, after migrating from the blood into the airways, a hyporeactive eosinophil remains hyporeactive to certain stimuli but is still capable of regaining and even increasing its functional responsiveness when exposed to cytokines. Given the constant secretion of cytokines in chronic inflammation, this observation might have important implications for the understanding of eosinophil cell biology in inflammation.
Is priming part of a broader concept of cell biology? The data discussed above suggest that priming is an inherent property of almost all inflammatory cells (possibly even all of them) and can be mediated by a myriad of ubiquitous soluble and insoluble factors. However, inflammatory-cell priming is not a well-defined, static cellular status but would be better regarded as a transient functional phase. This priming phase might be followed either by complete cellular activation and desensitization or by depriming when appropriate stimuli are absent and priming agents are lost. Further, within an inflammatory microenvironment, hyporeactive desensitized cells can undergo repriming (Fig. 1). The ability of inflammatory cells to undergo a cycle of priming, depriming and repriming reveals a previously unrecognized plasticity of inflammatory cells that is likely to be determined by the local microenvironment and that, in turn, dictates the course of inflammation.
Priming is associated with morphological, physical and phenotypic alterations of the cell. However, these alterations only partially overlap with the functional alterations (during the initial repriming process of a naive cell): they dissociate when cells become desensitized, deprimed or reprimed. Thus, the functional phenotype of inflammatory cells cannot be predicted from its surroundings nor from its morphological, physical and immunocytological characteristics. Because priming represents an essentially dynamic process, the concepts of cellular heterogeneity and priming are inaccurate and should be replaced by Ôcellular plasticityÕ for the following reasons. First, whereas the term heterogeneity implies the existence of welldefined and static subpopulations, cellular plasticity suggests that the observed differences are the result of an ongoing modulation of cell density, surface expression and function. Second, cellular plasticity takes into account the fact that cells should not be regarded as static biological units but rather as highly dynamic elements that constantly adapt to a given in vivo situation, defined by the surrounding microenvironment (made up of neighbouring cells, extracellular-matrix proteins and locally released mediators). Third, cellular plasticity accounts for the fact that inflammatory cells can adopt hyper-, poorly- and non-reactive functional states. Last, cellular plasticity is not restricted to the functional heterogeneity but also accounts for non-functional alterations, such as changes in morphological, physical and phenotypic features. Taken together, both priming and the heterogeneity of a given cell type are specific aspects of a broader concept in cell biology that also includes other cellular properties, such as depriming and repriming. Approaching inflammatory cells from the viewpoint of cellular plasticity might provide a better understanding of the pathogenesis of inflammatory disease and could assist in the development of novel therapeutic strategies.
We thank N. Kroegel for reviewing the manuscript. Our work was supported by the Bundesministerium fŸr Bildung und Forschung (01ZZ9602) and three grants by the Deutsche Forschungsgemeinschaft (Kr 956/2-1, 01KC8906/1 and Br 1949/1-1).
Claus Kroegel ([email protected]), Martin Foerster, Daniela HŠfner, Reinhard Grahmann and Ruedi Braun are at the Pneumology, Department IV, Medical University Clinics, Friedrich-Schiller University, Erlanger Allee 101, D-07747 Jena, Germany; Jane Warner is at the School of Biological Sciences, University of Southampton, Southampton, UK SO16 7PX.
References 1 Johnston, R.B. et al. (1981) Priming of macrophages for enhanced oxidative metabolism by exposure to proteolytic enzymes. J. Exp. Med. 153, 1678Ð1683 2 Miyagawa, H. et al. (1990) Density distribution and density conversion of neutrophils in allergic subjects. Int. Arch. Allergy Appl. Immunol. 93, 8Ð13 3 Ochensberger, B. et al. (1999) Regulation of cytokine expression and leukotriene formation in human basophils by growth factors, chemokines, and chemotactic agonists. Eur. J. Immunol. 29, 11Ð22
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4 Garraud, O. et al. (1997) Induction of parasite antigen-specific antibody responses in unsensitized human B cells is dependent on the presence of cytokines after T cell priming. J. Immunol. 159, 4793Ð4798 5 Koenderman, L. et al. (1996) Eosinophil priming by cytokines, from cellular signal to in vivo modulation. Eur. Respir. J. 9 (Suppl. 22), 119sÐ125s 6 Kroegel, C. et al. (1998) Eosinophil priming and migration in idiopathic pulmonary fibrosis. Eur. Respir. J. 11, 1Ð3 7 Kroegel, C. et al. (1994) Pulmonary immune cells in health and disease. The eosinophil leukocyte. Part I. Eur. Respir. J. 7, 519Ð543 8 Weller, P.F. (1991) The immunobiology of eosinophils. New Engl. J. Med. 324, 1110Ð1118 9 Silberstein, D.S. et al. (1989) Hemopoietins for eosinophils. Glycoprotein hormones that regulate the development of inflammation in eosinophila-associated disease. Hematol. Oncol. Clin. North Am. 3, 511Ð533 10 Takafuji, S. et al. (1991) IL-3 and IL-5 prime normal human eosinophils to produce leukotriene C4 in response to soluble agonists. J. Immunol. 147, 3855Ð3861 11 Kita, H. et al. (1992) Release of granule proteins from eosinophils cultured with IL-5. J. Immunol. 146, 629Ð635 12 Shindo, K. et al. (1996) Priming effect of platelet activating factor on leukotriene C4 from stimulated eosinophils of asthmatic patients. Thorax 51, 155Ð158 13 Chocrane, C.G. (1987) The enhancement of inflammatory injury. Am. Rev. Respir. Dis. 136, 1Ð2 14 Anwar, A.R.F. et al. (1993) Adhesion to fibronectin prolongs eosinophil survival. J. Exp. Med. 177, 839Ð834 15 Nagata, M. et al. (1995) Eosinophil adhesion to vascular cell adhesion molecule-1 activates superoxide anion generation. J. Immunol. 155, 2194Ð2202 16 Liles, W.C. et al. (1995) Cross-linking of CD18 primes human neutrophils for activation of the respiratory burst in response to specific stimuli, implications for adhesion-dependent physiological responses in neutrophils. J. Leukocyte Biol. 56, 690Ð697 17 Steeber, D.A. et al. (1997) Ligation of L-selectin through conservative regions within the lectin domain activates signal transduction pathways and integrin function in human, mouse, and rat leukocytes. J. Immunol. 159, 952Ð963 18 Dvorak, A.M. et al. (1990) in Megakaryocytes, platelets, macrophages and eosinophils (Vol. 2), pp. 237Ð344, Plenum 19 Yukawa, T. et al. (1989) Density heterogeneity of eosinophil leukocytes, induction of hypodense eosinophils by platelet activating factor. Immunology 681, 40Ð43 20 Kroegel, C. et al. (1993) Ultrastructural characterization of platelet activating factor-stimulated human eosinophils from subjects with asthma. Clin. Sci. 84, 391Ð399
21 Zeck-Kapp, G. et al. (1995) Mechanisms of human eosinophil activation by complement protein C5a and platelet activation factor, similar functional responses are accompanied by different morphological alterations. Allergy 50, 34Ð47 22 Hansel, T.T. et al. (1992) Induction and function of eosinophil intercellular adhesion molecule-1 and HLA-DR. J. Immunol. 149, 2130Ð2136 23 Berends, C. et al. (1997) Inhibition of PAF-induced expression of CD11b and shedding of L-selectin on human neutrophils and eosinophils by the type IV selective PDE inhibitor, rolipram. Eur. Respir. J. 10, 1000Ð1007 24 Luttmann, W. et al. (1996) Activation of human eosinophils by interleukin-13. Induction of CD69 surface antigen, its relationship to messenger RNA expression and promotion of cellular viability. J. Immunol. 157, 1678Ð1683 25 Kroegel, C. et al. (1994) Blood and bronchoalveolar eosinophils in allergic subjects after segmental antigen challenge. Surface phenotype, density heterogeneity and prostanoid production. J. Allergy Clin. Immunol. 93, 725Ð734 26 Sedgwick, J.B. et al. (1992) Comparison of airway and blood eosinophil function after in vivo antigen challenge. J. Immunol. 149, 3710Ð3718 27 Prin, L. et al. (1984) Heterogeneity of human eosinophils. II. Variability of respiratory burst activity related to cell density. Clin. Exp. Immunol. 57, 735Ð742 28 Shult, P.A. et al. (1988) The presence of hypodense eosinophils and diminished chemiluminescence response in asthma. J. Allergy Clin. Immunol. 81, 429Ð435 29 Kauffman, H.F. et al. (1987) Leukotriene C4 production by normaldensity and low-density eosinophils of atopic individuals and other patients with eosinophilia. J. Allergy Clin. Immunol. 79, 611Ð619 30 Hodges, M.K. et al. (1988) Heterogeneity of leukotriene C4 production by eosinophils from asthmatic and normal subjects. Am. Rev. Respir. Dis. 138, 799Ð804 31 Silberstein, D.S. et al. (1986) Enhancement of human eosinophil cytotoxicity and leukotriene synthesis by biosynthetic (recombinant) granulocyte-macrophage colony-stimulating factor. J. Immunol. 137, 3290Ð3294 32 Daniels, R.H. et al. (1992) Recombinant human monocyte IL-8 primes NADPH-oxidase and phospholipase A2 activation in human neutrophils. Immunology 75, 157Ð163 33 Ochensberger, B. et al. (1995) IgE-independent interleukin-4 expression and induction of a late-phase of a leukotriene C4 formation in human blood basophils. Blood 86, 4039Ð4049 34 Kitchen, E. et al. (1996) Demonstration of reversible priming of human neutrophils using platelet-activating factor. Blood 88, 4330Ð4337 35 Kroegel, C. et al. (1993) in Asthma Ð Immunopathology und Immunotherapy (Kummer, F., ed.), pp. 34Ð54, Springer
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Putting priming into perspective – from cellular heterogeneity to cellular plasticity Claus Kroegel, Martin Foerster, Daniela Häfner, P. Reinhard Grahmann, Jane A. Warner and Ruedi Braun The concept of priming is widely used in cell biology and has come to
U
nder certain in vivo and mediators, cell viability, adhesion, and mean the functional enhancement of in vitro conditions, the funcchemotaxis8,9. Similar spectra of cellular a given cell by cytokines. ÔPrimedÕ function enhanced by priming have also tion of inflammatory cells cells have a number of other cellular been observed for macrophages, neutrophils can be enhanced. This has and basophils. attracted much attention, as it explains the alterations, although the discrepancy between the high agonist conrelationship between functional and centrations required to elicit a cell response phenotypical diversity has not been Priming is achieved by soluble mediators in vitro and the relatively low concentrations Originally, priming was associated with the achieved in vivo. established. Here, Claus Kroegel and action of cytokines, and several in vitro studThe ability of cells to adopt an increased colleagues discuss the dynamic ies have shown that culturing eosinophils in functional status under certain conditions or the presence of interleukin 3 (IL-3), IL-5, in a defined environment is referred to as nature of inflammatory-cell granulocyteÐmacrophage colony-stimulating ÔprimingÕ. In addition to the functional varipriming, which might be part of a factor (GM-CSF), tumour necrosis factor a ability, priming has been closely associated broader means of comprehending (TNF-a) or interferon g makes the cell funcwith morphological, physical and phenotionally hyperactive8Ð10. However, this concept typical changes of the cell, providing the cell function in disease. needs to be modified in several ways. basis for the concept of cellular heterogeneity. First, in addition to their priming action, Although inflammatory-cell heterogeneity might be due largely to the site-specific blending of factors generated cytokines have also been shown to activate cellular effector functions within the local microenvironment, the relationship between prim- fully, including degranulation and other cellular responses11. Second, ing and other cellular alterations is still uncertain at present. There- mediators and agents that were known to activate eosinophils, such fore, this article brings together the various aspects of priming and as platelet-activating factor (PAF), complement factor 5a (C5a) and the chemotactic peptide N-formylmethionylphenylalanine (fMLP), discusses their relationship with other features of cellular diversity. have also been shown to prime eosinophils when applied at suboptimal (picomolar) concentrations12. Third, cell-permeable analogues What exactly is priming? of second messengers, phorbol ester, the calcium ionophore A23187, Priming is a feature of different cell types zymosan-activated serum and bacterial lipopolysaccharides have all The original concept of priming dates back to the observation by been shown to prime leukocytes13. Johnston et al.1 that macrophages show an enhanced oxidative burst The above findings indicate that the effect of a given soluble when exposed to proteases in vitro. However, priming is not re- agent (e.g. priming or activating) is determined by its actual concenstricted to macrophages but has also been observed in neutrophils2, tration rather than by its chemical nature, although other factors basophils3, lymphocytes4 and eosinophils5. Thus, priming represents such as the presence of cosecreted mediators, adhesive interactions a general phenomenon and is a functional option for many, if not all, with neighbouring cells or with intercellular matrix proteins might cell types. As eosinophils appear to respond to priming agents in a also play a role14,15. Therefore, the original concept that soluble very pronounced way, much of the information about priming was agents can be exclusively divided into priming and activating agents is not justified and should be abandoned. obtained with this cell type5Ð8.
Various cellular effector mechanisms are sensitive to priming
Priming by engagement of adhesion molecules
Most experiments dealing with priming assess only one or, occasionally, two cellular responses. However, priming is not restricted to a single cell function but covers myriad responses that a given cell is capable of performing. In the case of eosinophils, cellular properties induced by priming include respiratory burst, toxicity to host tissue and invading parasites, degranulation, formation of lipid
Eosinophils from allergic donors generate significantly more superoxide radicals in response to fMLP stimulation after adhering to a surface coated with vascular cell adhesion molecule 1 (Ref. 16). Monoclonal anti-CD18 antibodies prevented this adhesion-dependent priming effect, suggesting that this b2-integrin subunit is involved. Moreover, activation of the respiratory burst in human blood
0167-5699/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
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neutrophils exposed to fMLP, TNF-a or GM-CSF could only be achieved by crosslinking CD18 on the surface of the cells16. Another recent finding suggests that engagement of the L-selectin adhesion receptor serves as a priming signal, promoting subsequent firm cellto-cell adhesion17. These results clearly show that certain adhesion molecules are capable of priming inflammatory effector cells, although their mode of action is not fully understood.
How does priming relate to function and to other cellular alterations? Priming is associated with morphological alterations Eosinophils from patients with eosinophilia are morphologically distinct from cells from healthy subjects7Ð10. The changes include the appearance of cytoplasmic vacuoles, alterations in cell size and shape, an increase in granule size, solubilization and loss of granular-core and matrix proteins, and an increase in smooth tubules and vesicles18. Intracellular morphological alterations observed in vivo can also be induced by exposing eosinophils to cytokines, C5a or PAF in vitro19Ð21, suggesting that morphological heterogeneity is a general feature of inflammatory cells and is caused by inflammatory mediators.
Priming is related to physical cell changes Blood from patients with eosinophilia-associated diseases contains a population of eosinophils that segregates at a lower buoyant density when centrifuged through Percoll or Metrizamide gradients; these are known as ÔhypodenseÕ, Ôlight-densityÕ or Ôlow-densityÕ eosinophils7,8. In addition, eosinophils recovered from both pleural and bronchoalveolar spaces contain a high proportion of hypodense cells. Hypodense eosinophils are larger than normal ones, contain fewer partially lucent granules and have increased levels of both adhesion molecules and function5, revealing a remarkable resemblance to in-vitro-induced hypodense eosinophils9. Furthermore, the hypodense subpopulation often shows a variably enhanced functional response, either spontaneously or following stimulation in vitro. Moreover, normodense blood eosinophils (i.e. those with normal density) shift towards the hypodense subtype when cultured in the presence of IL-3, IL-5, GM-CSF, C5a, PAF or fMLP at both activating and priming concentrations5,9,19.
Priming is accompanied by phenotypic changes Inflammatory cells have the inherent ability to modify their expression of surface receptors. In eosinophils, for instance, a number of membrane antigens (including the complement receptors and adhesion molecules) are constitutively expressed and might be upregulated on exposure to mediators22. By contrast, other constitutively expressed surface antigens (such as L-selectin) are shed during eosinophil activation and transendothelial migration23. Finally, certain surface antigens are newly synthesized and expressed in primed or activated eosinophils, including class II proteins of the major histocompatibility complex, CD4, intercellular adhesion molecule 1
(ICAM-1) and IL-2 receptor (IL-2R)8,22Ð24. Again, these changes typically occur in vitro after the cells are cultured with cytokines known to prime eosinophils, such as IL-3, IL-5, IL-13 and GM-CSF7,20Ð23. A similarly altered phenotype can be observed in eosinophils recovered from bronchoalveolar lavage (BAL) or the sputum or blood of patients with chronic inflammatory diseases22,25Ð27. For instance, BAL and sputum eosinophils obtained from patients with atopic asthma and eosinophilic pneumonia express ICAM-1, but L-selectin expression is lower than on blood cells. Furthermore, in both diseases, the BAL and sputum eosinophils show an upregulation of the b2 integrins CD11b and CD11c, as well as the leukocyte function-associated antigen 3 (LFA-3; CD58). Taken together, these data indicate that characteristic phenotypic changes are associated with the functional changes.
Hypodense cells that are activated are not always primed Hypodense cells obtained from asthmatics produced fewer reactiveoxygen metabolites in response to zymosan than normodense cells, as assessed by nitroblue tetrazolium reduction and chemoluminescence27,28. In addition, hypodense blood eosinophils from asthmatics produced only half the amount of leukotriene C4 (LTC4) that normal cells did29. Further, BAL eosinophils from asthmatic subjects extracted 18 h after an allergen challenge had a reduced capacity to generate both prostanoids and leukotrienes (e.g. prostaglandin D2, thromboxane A2 and LTC4) than blood eosinophils when stimulated with PAF (Ref. 25). Similarily, peripheral-blood eosinophils from asthmatics synthesized less LTC4 than cells isolated from normal subjects30. In a study that compared blood and BAL eosinophils obtained from asthmatics 48 h after allergen provocation, airway eosinophils generated significantly more superoxide on activation with fMLP than cells from normal subjects but failed to do so when activated with PAF, phorbol 12-myristate 13-acetate or the Ca21 ionophore A23187 (Ref. 26). These data suggest that hypodense airway eosinophils expressing certain surface antigens associated with priming or activation, in addition to having enhanced functional properties, reveal an unaltered or a reduced functional capacity that is stimulus dependent.
Is priming a singular event? Priming is a reversible cellular process There is mounting evidence suggesting that the enhanced functional status of a primed cell spontaneously returns to the prepriming levels (depriming). For instance, human basophils stimulated with C5a only induced the production of IL-4, IL-13 and LTC4 after pretreatment with IL-3, IL-5 or GM-CSF. In addition, the priming of eosinophils with GM-CSF reached a maximum 60 min after the agonist was added and declined thereafter31. A similar pattern has been observed with several other cells32, although the time course can depend on the priming agent employed. Whereas the priming effect of IL-3 persisted for more than 18 h (Ref. 33), the action of both IL-5 and GM-CSF disappeared within a few hours3,33. These observations suggest that priming is a reversible process and that the biological effect of priming depends on the time lag between exposure to the priming agent and the activating stimulus. The data further stress
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Fig. 1. Simplified schematic representation of post-mitotic eosinophil differentiation. After differentiation, maturation and release from the bone marrow, the cell has normal density (is ÔnormodenseÕ) and is functionally passive (1). In the absence of disease, the cell will unselectively migrate into any tissue and will eventually undergo apoptosis without having performed its effector functions (ÔageingÕ) (2). However, during disease (e.g. hypereosinophilic syndrome, parasitic infection or allergy), systemically available cytokines will initiate cellular priming (3). Depending on the type, course and severity of the underlying disorder, several options are available to the circulating cell. When appropriate stimuli are absent and priming agents are lost, the priming phase can be followed by depriming, leading to an unreactive, passive cell that will eventually die (4). When primed circulating cells passing through an area of ongoing inflammation, local mediators can trigger immediate transendothelial migration. In the continued presence of priming stimuli (adhesion molecules, matrix proteins) the migrating cell retains its functionally active state but, at the same time, adopts a hypodense phenotype and undergoes further morphological changes (5). As the cell approaches the inflammatory focus, increasing mediator concentrations induce full cellular activation and the release of mediators and other effector molecules. Complete cellular activation is followed by transient desensitization, leaving the hypodense cell in a functionally unresponsive state (6). However, if local cytokines and other mediators continue to be present within an inflammatory microenvironment, hyporeactive desensitized cells can be reprimed, regaining partial or complete effector functions (7). Finally, if no additional mediators are offered, the cells remain within the circulation, slowly adopting a hypodense phenotype (8) and transmigrating non-selectively into any tissue. Owing to the raised functional reactivity of the cell, effector molecules are released after interaction with extracellular-matrix proteins and other receptors. Abbreviations: C5a, complement factor 5a; GM-CSF, granulocyteÐmacrophage colony-stimulating factor; IL-3, interleukin 3; LTB4, leukotriene B4; PAF, platelet-activating factor. Modified from Ref. 35.
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the importance of the timing and sequence of biologically active molecules for inflammatory cell performance.
Cells are susceptible to repeated priming Although priming is generally seen as a one-off, irreversible process, there is evidence that this may not always be true. It is well known that fMLP stimulates superoxide-anion release by neutrophils, leading to cellular shape changes, and inducing CD11b-dependent cell binding to albumin-coated surfaces following priming with concentrations of PAF below the activation threshold. This priming effect spontaneously declines when fMLP is added 10 min after PAF and disappears altogether after 120 min34. Moreover, a PAF-receptor antagonist added 10 min into the PAF priming period accelerated depriming of the neutrophils. More importantly, cells that had spontaneously deprimed after PAF exposure retained their capacity to be fully reprimed by subsequent addition of PAF. These observations imply that a cell can undergo priming repeatedly.
Priming can restore the function of hyporeactive cells Hypodense BAL eosinophils obtained during the late asthmatic response (18 h after endobronchial allergen challenge) synthesized less lipid mediators, either spontaneously or following stimulation, than blood cells from the same patient25. However, when the cells were pretreated with non-activating concentrations of GM-CSF for 60 min in vitro before stimulation with PAF or the Ca21 ionophore A23187, the cells regained a hyper-responsive phenotype35 (as assessed by thromboxane synthesis and LTC4 release). At the same time, their density decreased further and the expression of PAF receptors increased by more than 50%. These data suggest that, after migrating from the blood into the airways, a hyporeactive eosinophil remains hyporeactive to certain stimuli but is still capable of regaining and even increasing its functional responsiveness when exposed to cytokines. Given the constant secretion of cytokines in chronic inflammation, this observation might have important implications for the understanding of eosinophil cell biology in inflammation.
Is priming part of a broader concept of cell biology? The data discussed above suggest that priming is an inherent property of almost all inflammatory cells (possibly even all of them) and can be mediated by a myriad of ubiquitous soluble and insoluble factors. However, inflammatory-cell priming is not a well-defined, static cellular status but would be better regarded as a transient functional phase. This priming phase might be followed either by complete cellular activation and desensitization or by depriming when appropriate stimuli are absent and priming agents are lost. Further, within an inflammatory microenvironment, hyporeactive desensitized cells can undergo repriming (Fig. 1). The ability of inflammatory cells to undergo a cycle of priming, depriming and repriming reveals a previously unrecognized plasticity of inflammatory cells that is likely to be determined by the local microenvironment and that, in turn, dictates the course of inflammation.
Priming is associated with morphological, physical and phenotypic alterations of the cell. However, these alterations only partially overlap with the functional alterations (during the initial repriming process of a naive cell): they dissociate when cells become desensitized, deprimed or reprimed. Thus, the functional phenotype of inflammatory cells cannot be predicted from its surroundings nor from its morphological, physical and immunocytological characteristics. Because priming represents an essentially dynamic process, the concepts of cellular heterogeneity and priming are inaccurate and should be replaced by Ôcellular plasticityÕ for the following reasons. First, whereas the term heterogeneity implies the existence of welldefined and static subpopulations, cellular plasticity suggests that the observed differences are the result of an ongoing modulation of cell density, surface expression and function. Second, cellular plasticity takes into account the fact that cells should not be regarded as static biological units but rather as highly dynamic elements that constantly adapt to a given in vivo situation, defined by the surrounding microenvironment (made up of neighbouring cells, extracellular-matrix proteins and locally released mediators). Third, cellular plasticity accounts for the fact that inflammatory cells can adopt hyper-, poorly- and non-reactive functional states. Last, cellular plasticity is not restricted to the functional heterogeneity but also accounts for non-functional alterations, such as changes in morphological, physical and phenotypic features. Taken together, both priming and the heterogeneity of a given cell type are specific aspects of a broader concept in cell biology that also includes other cellular properties, such as depriming and repriming. Approaching inflammatory cells from the viewpoint of cellular plasticity might provide a better understanding of the pathogenesis of inflammatory disease and could assist in the development of novel therapeutic strategies.
We thank N. Kroegel for reviewing the manuscript. Our work was supported by the Bundesministerium fŸr Bildung und Forschung (01ZZ9602) and three grants by the Deutsche Forschungsgemeinschaft (Kr 956/2-1, 01KC8906/1 and Br 1949/1-1).
Claus Kroegel ([email protected]), Martin Foerster, Daniela HŠfner, Reinhard Grahmann and Ruedi Braun are at the Pneumology, Department IV, Medical University Clinics, Friedrich-Schiller University, Erlanger Allee 101, D-07747 Jena, Germany; Jane Warner is at the School of Biological Sciences, University of Southampton, Southampton, UK SO16 7PX.
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