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- Material and methods
Background: The study aimed to investigate whether CD69 expression on granulocytes is subject to specific regulation by inflammatory mediators, and, if so, to identify these factors in relation to eosinophil activity markers such as the EG2 epitope and ECP release.
Methods: Peripheral blood leukocytes from healthy donors were used. The surface and intracellular distribution of CD69 was investigated with a whole-blood cell-membrane permeabilization technique, the FOG method, and flow cytometry. In vitro stimulation was performed with GM-CSF, IL-5, IL-5 plus eotaxin, LPS, and fMLP.
Results: A preformed intracellular pool of CD69 was demonstrated in both eosinophils and neutrophils, but not in monocytes. Almost no resting eosinophils, neutrophils. or monocytes expressed CD69 on the cell surface. However, in vitro stimulation with selected stimuli increased the proportion of CD69-positive eosinophils to various extents, with GM-CSF being the most and fMLP the least efficient stimulus. The neutrophils did not respond under these conditions. Increased expression of the EG2 epitope and initiation of degranulation preceded CD69 upregulation.
Conclusions: Eosinophils and neutrophils from healthy donors have a preformed intracellular pool of CD69, which is mobilized on the cell surface on eosinophils, but not on neutrophils, to various extents by selected stimuli. Monocytes, however, do not have a preformed intracellular pool of CD69. Our data indicate that a kinetic order exists among the EG2 expression, the degranulation process, and CD69 upregulation. Due to a quantitative, rather then a qualitative, upregulation of CD69 by stimuli associated with both allergic and bacterial inflammation, CD69 may be a potential activity marker of clinical value.
Eosinophils are associated with a number of diseases, especially asthma, atopic dermatitis, and helminthic parasite infection ( 1). Histopathologic studies have shown an increased influx of eosinophils to the allergic site of inflammation with subsequent tissue damage, and it is now generally believed that eosinophilic accumulation is a characteristic feature of allergic inflammation ( 2, 3). Neutrophils, on the other hand, are recruited into sites of acute bacterial infection, but that does not exclude the possibility of neutrophilic contribution to the patho-physiology in allergic diseases ( 4–6).
No single granulocyte activity marker has been described that alone provides a useful tool for monitoring asthma or allergic inflammation. Increased number of eosinophils has been used as a monitoring marker in the management of asthma, and it has been reported that the total number of peripheral blood eosinophils (PBE) correlates with disease severity ( 7, 8). However, not only the number of PBE but also their state of activation could reflect an ongoing allergic inflammatory process ( 9, 10). The expression of the EG2 epitope on intracellular eosinophil cationic protein (ECP) is one among several markers that have been used to estimate eosinophil activity both in vitro and in vivo ( 11–14). Another marker used to identify eosinophil activation, ECP is released during the allergic inflammatory process ( 15–17).
The antigen CD69 is a 60-kDa glycoprotein composed of two subunits of 27 and 33 kDa, respectively ( 18). The ligand of CD69 is still to be identified, but a membrane-bound molecule is more likely than a soluble factor, since cross-linking of CD69 is required for optimal stimulation ( 19, 20). Surface expression of CD69 has been observed on various cells, such as activated T and B cells ( 18, 21, 22), natural killer (NK) cells ( 23), monocytes ( 24), neutrophils ( 25), and platelets ( 26). However, PBE from healthy subjects do not express a significant amount of CD69 on the cell surface ( 27). On the other hand, the main body of eosinophils recovered from bronchoalveolar lavage (BAL) in patients with eosinophilic pneumonia ( 28), as well as with asthma ( 27), express this antigen on the cell surface. Collectively, these data indicate that CD69 can be expressed on eosinophils recruited into inflammatory/infectious sites.
Given that the surface expression of CD69 on human eosinophils may vary in relation to the state of activation, our present study focused on the in vitro regulation of CD69 in peripheral blood eosinophils, neutrophils, and monocytes. The specific aim was to investigate whether CD69 expression is subject to specific regulation by inflammatory mediators, and, if so, to identify these factors in relation to eosinophil and neutrophil function.
- Top of page
- Material and methods
In this study, the regulation of CD69 in human peripheral blood eosinophils and neutrophils from healthy individuals was investigated in relation to in vitro activation with GM-CSF, IL-5, IL-5 plus eotaxin, LPS, and fMLP. We were able to demonstrate a preformed intracellular pool of CD69 in both eosinophils and neutrophils. The intracellular pool of CD69 was mobilized on the cell surface on eosinophils to different degrees by selected stimuli, with GM-CSF being the most efficient and fMLP the least efficient stimulus. In contrast, CD69 in neutrophils was not mobilized on the surface under these experimental conditions.
Previous reports have identified CD69 on different cell types, including activated T cells, B cells, NK cells, monocytes, neutrophils, and platelets ( 18, 21–25). In our present study, we demonstrated that almost all PBE and PBN have a preformed intracellular pool of CD69, and approximately 5% of resting PBE and 1% of resting PBN from healthy individuals express the antigen on the surface. These figures are in line with previously reported data ( 25, 34). However, it has been proposed that eosinophils recovered from BAL in patients with asthma ( 27) and eosinophilic pneumonia ( 28, 35) do express cell-surface CD69. Furthermore, stimuli such as GM-CSF, IL-3, IL-4, IL-5, IL-13, and IFN-γ can upregulate CD69 on eosinophils in vitro ( 27, 28, 34, 36, 37).
Eosinophils are derived from multipotent stem cells in the bone marrow in the presence of eosinophilopoietic cytokines such as IL-5 and GM-CSF. Besides promoting the terminal differentiation of the eosinophil precursors, these cytokines enhance different aspects of eosinophil function, such as chemotaxis, prolonged survival, and activation ( 38). When measuring the surface expression of CD69 on eosinophils and neutrophils after in vitro stimulation with cytokines such as GM-CSF and IL-5, we observed an increased expression of CD69 on eosinophils, but not on neutrophils. In another series of experiments, we detected a decrease in the intracellular pool of CD69 in PBE after stimulation with GM-CSF, whereas the preformed pool in neutrophils was not significantly changed. However, we noted a significantly increased number of surface CD69 on neutrophils, an effect which was less pronounced than on eosinophils. These results indicate that different mechanisms regulate the cell-surface mobilization of CD69 in eosinophils than in neutrophils.
To investigate whether additional stimuli, more related to infections, also may upregulate CD69, we used the bacteria-related products LPS and fMLP. Approximately 50% of the eosinophils responded with CD69 surface upregulation after LPS stimulation, and approximately 17% responded after fMLP stimulation. These results indicate that stimuli related to bacterial infection are less efficient than more eosinophil-specific stimuli, in the context of CD69 upregulation properties, and that the observed differences between these families of stimuli are more quantitative, than qualitative in nature. When we used LPS as stimulus, a significantly lower (P<0.01) degree of CD69 expression was measured on purified eosinophils than on eosinophils in mixed leukocytes. This might be due to a cascade effect whereby LPS activates the monocytes, which produce a factor or factors that promote CD69 upregulation. It is plausible that such a factor is GM-CSF, which is released upon LPS stimulation ( 39).
The neutrophils did not respond with CD69 upregulation under the experimental conditions used in the present study. The function of CD69 in neutrophils is not fully understood, and our results do not exclude the possibility that CD69 may be mobilized on the cell surface on neutrophils under different in vitro conditions.
In contrast to De Maria et al.'s report that CD69 is expressed on all purified monocytes ( 24), our results show that the main body of nonstimulated peripheral blood monocytes, without prior steps of purification, neither have a preformed intracellular pool nor express CD69 on the cell surface. The reason for this conflicting finding is unclear, but previous studies have shown that platelets, which are known to express CD69 ( 26), adhere to monocytes under different ex vivo conditions ( 40, 41). Our presented data demonstrate that purification with gradient centrifugation results in a higher number of CD61-positive monocytes than nonseparated monocytes, indicating that adherence of platelets to monocytes occurs during the separation process. Since platelets constitutively express CD69, ex vivo adherence of platelets to monocytes may be a plausible explanation of previous reports of CD69-positive monocytes.
In this study, we compared the expression of CD69 with other activity markers, the intracellular expression of the EG2 epitope on ECP, and the release of ECP. Stimulation with GM-CSF resulted in a pronounced upregulation of CD69 that peaked late during the actual incubation period. This kinetic pattern was in contrast to the EG2 and ECP pattern, which showed a more gradual and moderate increase, without any pronounced peak during late incubation. These results indicate that increased expression of the EG2 epitope, and the ECP release, precede the main upregulation of CD69, upon GM-CSF stimulation. The eventual impact of these findings is not clear, but may indicate that a kinetic order exists between degranulation and CD69 upregulation, and that the physiologic role of surface CD69 is primary after the initiation of the degranulation process. In line with this hypothesis is the notion that ligation of anti-CD69 induces apoptosis in eosinophils ( 42). One may hypothesize that a faulty upregulation of CD69 may play an important role in defect apoptosis, which may lead to prolonged survival of inflammatory cells in a state that permits degranulation. The consequence of this might be increased tissue damage, as observed in asthma and lung fibrosis.
In summary, our results show that both eosinophils and neutrophils have a preformed intracellular pool of CD69, and that hardly any eosinophils and neutrophils in the circulation of healthy individuals express CD69 on the cell surface. The intracellular pool can be mobilized on the surface of eosinophils, but not of neutrophils, by GM-CSF, IL-5, and LPS and, to a lesser extent, by the bacteria-related product fMLP. This indicates that a more quantitative than qualitative difference in CD69 upregulation abilities among stimuli exists. Moreover, our data indicate that a kinetic order exists between the degranulation process and CD69 upregulation, and that CD69 is an activity marker that appears later during activation than the increased expression of the EG2 epitope and the ECP release.
For further delineation of the role of CD69 on PBE and PBN in allergic disorders as compared to bacterial infections, analysis of CD69 expression on PBE and PBN from patients with inflammatory processes of different magnitude and nature would seem to be important.