The border-crossing behavior of eosinophils and neutrophils in the lung - Summary & Conclusions

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The border-crossing behavior of eosinophils and neutrophils in the lung

Zuurbier, A.E.M.

Publication date

2001

Link to publication

Citation for published version (APA):

Zuurbier, A. E. M. (2001). The border-crossing behavior of eosinophils and neutrophils in the

lung.

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Summary & Conclusions

Migration of eosinophils across epithelial monolayers

In the late phase of allergic asthmatic reactions massive amounts of eosinophils infiltrate the lungs and cause damage to the tissue (Chapter I ). In this study we have tried to contribute to the elucidation of the mechanism that is at the basis of this infiltration. To investigate this, we have used an in-vitro transmigration model, in which leukocyte migration through cellular monolayers can be analyzed under various conditions. The migration of eosinophils and neutrophils across endothelial monolayers

in vitro has already been studied extensively (Chapter 1 ). but less is known about migration of these

leukocytes across lung epithelial monolayers. Therefore, we have investigated the effect of putative participants in allergic asthmatic inflammation on the migration of eosinophils across (primary) bronchial epithelial monolayers.

We have studied the effect of platelet-activating factor (PAF) on eosinophil transepithelial migration in vitro, and found that PAF synergistically enhances eosinophil migration across bronchial epithelial monolayers (Chapter 2). Our results indicate that PAF is not only a chemoattractant but also a priming agent for eosinophils and that the enhancement of eosinophil transepithelial migration is partly due to the direct action of PAF on the epithelial cells. Binding of PAF to its receptors on bronchial epithelial cell membranes induces an transient increase in [Ca:+]j and triggers a cascade of

intracellular signals that leads to functional changes in the epithelial cells. These changes possibly include loosening of the intercellular junctions between adjacent epithelial cells, which favors passage of eosinophils. Neutrophil transepithelial migration is more rapid and efficient than eosinophil migration and it is not enhanced by PAF (Chapter 3). The latter observation can possibly be explained by the assumption that neutrophils or neutrophil-derived products, e.g. PAF, already trigger loosening of the intercellular junctions. Generally speaking, it can be concluded that epithelial cells do not form a passive barrier but play an active role in leukocyte transmigration.

Neutrophils have been described to participate in allergic inflammation in the lungs of asthmatic individuals (Chapter I ). From our in-vitro studies it appeared that addition of neutrophils to the upper compartment of the transmigration model stimulates C5a-driven migration ot eosinophils across confluent cellular monolayers (Chapter 3). Our results suggest that this stimulatory effect is due to maintenance of the C5a chemotactic gradient by neutrophil-mediated inactivation of C5a that has diffused into the upper compartment. This indicates that not only the presence but also the local concentration of chemoattractants is of vital importance for their function in inflammation.

In vivo. C5a acts as a chemoattractant and activator of both eosinophils and neutrophils and is

implicated in the inflammatory reaction in the lungs of allergic asthmatic patients. We hypothesize that //;

vim. invading neutrophils, which migrate ahead of eosinophils, neutralize C5a in the surrounding tissue

and vasculature, resulting in maintenance of the C5a chemotactic gradient, and hence, in enhanced attraction of eosinophils towards the site of allergic inflammation. Naturally, this is not the only function of neutrophils in allergic inflammation, because neutrophils produce and release sc\ cral kinds of inflammatory mediators and toxic agents (Chapter 1 ). which also affect the process of inflammation. In fact, the importance of the C5a-netitralizing capacity of neutrophils in lung inflammation remains to be determined, although this may be extremely difficult to determine in vivo.

Summary & Conclusions

Paracrine interaction between endothelial and epithelial cells

In allergic asthmatic inflammation, extravasation of granulocytes is followed by passage through the basement membrane and contact with a second cell type. e.g. epithelial cells in the lung. In an attempt to mimic this complex in-vivo situation, we de\ eloped an experimental model to investigate granulocyte migration in the context of lung inflammation, using monolayers of primary endothelial cells and primary bronchial epithelial cells simultaneously cultured on opposite sides of the same Transwell filter (the bi-layer system). We have specifically investigated the migration of neutrophils and eosinophils across these bi-layers and the production of cytokines and chemokines by these cellular bi-layers. The first results obtained with this model indicate that the endothelial and epithelial cell types indeed influence each other, resulting in stimulated production of cytokines, accompanied by enhanced neutrophil migration (Chapter 4). The transmigration of neutrophils in the bi-layer model was more efficient than in the single epithelial monolayer model, and at least equaled the migration in the single endothelial monolayer model. Several mechanisms may be involved in this phenomenon. We excluded a possible stimulating effect of enhanced neutrophil adhesion to the endothelial monolayer, of increased neutrophil motility after interaction with the ECM, and of physical contact between the endothelial and epithelial cells. Instead, a paracrine interaction between the epithelial and the endothelial cells seems to be implicated in the increase of neutrophil transmigration.

To study the effect of this paracrine interaction on endothelial and epithelial cells and to further investigate the roles of either cell type in the stimulated neutrophil migration across the bi-layers. we have analyzed the behavior of the endothelial and epithelial cells in a co-culture model. Endothelial and epithelial cells were simultaneously cultured in the same wells but on different surfaces, i.e. on the Transwell filter and on the bottom of the Transwell system, to prevent any physical contact. The paracrine interaction between the cell types was indeed confirmed by the observation that neutrophil transmigration is increased when endothelial cell and epithelial cell monolayers are co-cultured (Chapters 4 and 5).

As to the mediators invoked, we found (Figure I ) that cultured bronchial epithelial cells continuously produce IL-I (], which in turn stimulates endothelial cells to produce IL-8 and maintains epithelial cell IL-8 production through an autocrine loop (Chapter 4). IL-8 is a potent neutrophil

chemoattractant that is surely involved in the stimulatory effect on neutrophil migration in the bi-layer and the co-culture models. This participation of IL-8 in stimulated neutrophil migration was further supported bv the finding that eosinophil migration across bi-layers was not enhanced (data not shown), since IL-8 is a weak eosinophil chemoattractant. However, the entire effect of enhanced neutrophil migration cannot only be due to production of IL-8. because neutrophil migration across bi-layers was not enhanced in the absence of added chemoattractants in the lower compartment. The concentration of 1L-6, a

pro-inflammatory mediator that enhances neutrophil surv ival, was also enhanced in our migration models, due to stimulated production by the epithelial cells. This cytokine has been described to decrease cell-cell association between carcinoma cells, but whether or not I L-6 also affects the epithelial intercellular junctions and neutrophil migration remains to be elucidated.

Besides the described production of cytokines and chemokines. co-culture of endothelial cells and epithelial cells may also affect their extracellular make-Lip. which may in turn influence the passage of leukocytes. When the accumulated cytokines in the supernatants were removed by washing just before the start of the transmigration assay, we found that the spontaneous neutrophil migration. i.e. in the absence of chemoattractants. across co-cultured endothelial monolayers, was enhanced. This could be the result of a change in the expression of adhesion molecules on the co-cultured endothelial

cells. The cell-surface expression of ICAM-I, CD3 1 and ßr integrin was up-regulated, and the

ICAM-2 expression was down-regulated on co-cultured endothelial cells (Chapter 5). The expression of 1CAM-1. CD31. |i,-intcgnn and ICAM-2 may be regulated by IL-lß, in concert with other mediators present in the co-culture supernatant.

IL-I ß is most likely derived from the cultured bronchial epithelial cells (Chapter 4). Thus, in the bi-layer and co-culture models, epithelial-cell derived IL-lß undoubtedly plays a central role in the modulation of endothelial cell function, e.g. it enhances II.-8 production and regulates adhesion molecule expression (Fig. 1 ). However, whether or not IL-I [i is indeed the initiating factor in the stimulated neutrophil migration across the bi-laycrs remains to be established. Moreover, the mediators that are released by endothelial cells, possibly in response to IL-1 ß stimulation, and then activate epithelial cells, still need to be identified. A possible candidate for such an cndothelial-ccll derived mediator is PAF. This acctylphospholipid is produced by endothelial cells, and we have shown that it activates epithelial cells, leading to facilitated passage of eosinophils without having an effect on the expression of ICAM-1 (Chapter 2). I ICAM-2 T ICAM-1, (!,-integrin, CD31 IL-1ß IL-8 IL-6 i"i i i ? Endothelial cells

<Jr

Figure 1. Schematic representation of the paracrine interaction between endothelial and epithelial cells. The expression of VCAM-I was not stimulated on co-cultured or IL-I ß-treated endothelial cells (Chapter 5). VCAM-1 expression is up-regulated by allergy-related cytokines, such as IL-4 (Chapter 7), and it is especially implicated in eosinophil, but not neutrophil, transendothelial migration. Whether or not the lack of enhancement of eosinophil migration through bi-laycrs (data not shown) can indeed be ascribed to this "IL-I ß-like" activation state of the endothelial monolayer remains to be elucidated.

As yet, we did not detect any co-culture-induced effects on epithelial cells that may promote neutrophil transepithelial migration, although the migration across bi-layers and co-cultured epithelial monolayers was found to be increased in comparison to migration across single epithelial monolayers (Chapter 4). The expression of certain adhesion molecules on bronchial epithelial cells was not affected by co-culture with endothelial cells either. This cannot simply be explained by the argument that these cells already have an activated phenotypc, because we found that the cultured bronchial epithelial cells can be stimulated to produce IL-6 (Chapter 4). Possibly, modulation of expression of these molecules is not required for efficient leukocyte passage. Invading leukocytes do not need to cling to the epithelium like they do when interacting with endothelial cells in the circulation, i.e. surrounded by an environment with high shear stress. Moreover, epithelial cells are not involved in selective leukocyte recruitment or in

Summary & Conclusions

recruitment at particular sites in inflamed organs, because these ceils only encounter leukocytes that have already been recruited by the endothelium. On the contrary, invading leukocytes more or less encounter the epithelium on their way through the tissue to the site of inflammation, and the constitutive presence of these adhesion molecules may be enough to guide the leukocytes through the process of transepithelial migration. Nevertheless, epithelial cells have been shown to actively participate in granulocyte

transmigration (Chapter 2). In \ lew of the compact and thick aspect of the epithelium, changes induced on the intercellular junctions may be of more importance to leukocyte passage than changed expression of adhesion molecules. Also the activation state of the adhesion molecules may have been altered. These topics have not yet been thoroughly addressed.

Role of PECAM-1 in neutrophil and eosinophil extravasation

In the process of the characterization of "trans-bi-laycr" migration of neutrophils and eosinophils, we noted that neutrophil, but not eosinophil, PECAM-1 is implicated in transendothelial migration in

vitro, although both cell types express PECAM-1 at similar levels (Chapter 6). Our results suggest

that neutrophil PECAM-1 interacts with an, as yet unknown, ligand on endothelial cells. Interaction v\ ith this ligand could lead to activation of the neutrophils and result in enhanced migration. A potential candidate for such a ligand is avßj.

In eosinophil transendothehal migration, such an interaction between eosinophil PECAM-1 and an endothelial ligand is cither absent or plays a less dominant role. Besides this difference in PECAM-1 function, the migratory capacity of eosinophils and neutrophils differ in that neutrophils migrate more efficiently than eosinophils across either monolayers of endothelial cells or epithelial cells (Chapter 3). Moreover, we found that neutrophil, but not eosinophil, migration across endothelial-epithelial bi-layers is enhanced (data not shown). Taken together, these findings underscore the fact that neutrophils and eosinophils are differently equipped for their specific functions in inflammation. Each of these factors is probably implicated in the selectivity of leukocyte recruitment during inflammatory reactions.

Lung versus umbilical vein endothelial cells

Last but not least, we intended to investigate the migration across lung microvascular endothelial cells (LMVEC) as compared to migration across endothelial cells derived from umbilical veins (HUVEC) that arc routinely used for the study of transendothehal migration in vitro. We reasoned that specific features of LMVEC might contribute to the eosinophil and neutrophil recruitment as seen in allergic inflammation. We have tried to isolate and culture LMVEC, but we found that isolation of human LMVEC is extremely difficult (Chapter 7). Despite a great deal of effort, we did not succeed in isolating and cultivating human LMVEC. most probably due to our access to lung tissue only from lung cancer patients. The condition of the lung endothelial cells may have been insufficient for

in-vitro survival or proliferation from the very beginning.

Recently, human LMVEC derived from human material used for transplantation purposes have become commercially available, and this enabled us to study these cells after all (Chapter 7). Assessment of the surface expression of adhesion molecules on LMVEC and HUVEC revealed that the expression of ßrintegrins, PECAM-1 and ICAM-I is higher on LMVEC than on HUVEC, and

stimulated E-selectin, ICAM-I and VCAM-I expression on LMVEC, but not on HUVEC, whereas TNF-a potently stimulated E-selectin expression on HUVEC, but not on LMVEC.

In brief, we found that the expression of adhesion molecules on cultured endothelial cells derived from umbilical cord and lung tissue is not only different on resting cells but also differentK regulated on stimulated cells. This difference may even be greater when the cells arc imbedded in their natural environment, i.e. surrounded by locally produced cytokines and extracellular matrix proteins. The regulation of expression of adhesion molecules on endothelial cell lines is likely differently regulated as well. Findings obtained with cell lines were therefore routinely verified findings with early passages of primary cells.

Conclusions

In conclusion, we have shown that addition of an extra cell type, for instance neutrophils, endothelial cells or epithelial cells, to the in-vitro migration model affects the transmigration of leukocytes. This can be the result of an indirect effect on the environment, e.g. C5a inactivation by neutrophils, or a direct effect, e.g. a paracrine interaction between the cells. We have developed a new in-vitro transmigration model in which this paracrine interaction can be studied and found that it affects the secretion of cytokines, the expression of adhesion molecules, and possibly the intercellular junctions. Together, these changes, and maybe other co-culture-induced changes as well, result in augmented migration of neutrophils. //; vivo, eosinophils and neutrophils are also subject to several modulating factors, e.g. to cytokines, chemokines and contact with other cell types. Therefore, our results support the notion that eosinophil and neutrophil infiltration in the lungs of asthmatic individuals is the result of a complex interplay between various cell types with specific features, which in turn differ in their potency to modulate the inflammatory reaction. The major aim of future research should be to unravel the observed effects, especially concerning the changes inflicted upon the intercellular junctions, and to determine the significance of the divergent effects in allergic inflammation in vivo.