Article |
Address correspondence to Peter R. Kvietys, Program in Vascular Biology/Inflammation, Lawson Health Research Institute, 375 South Street, Room C210, London, Ontario, Canada N6A 4G5. Tel.: (519) 685-8300 ext. 77055. Fax: (519) 667-6629. E-mail: pkvietys{at}julian.uwo.ca
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Abstract |
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Key Words: ICAM-1; CD18; HUVEC; mice; NO
* Abbreviations used in this paper: CD18, cluster of differentiation-18; CLP, cecal ligation and perforation; EMSA, electrophoretic mobility shift assay; HUVEC, human umbilical vein endothelial cell; ICAM-1, intercellular adhesion molecule 1; I
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Introduction |
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The systemic inflammatory response appears to be a self-amplifying phenomenon generated by the activation of nuclear transcription factors by circulating cytokines. One transcription factor that is believed to be important in the systemic inflammatory response is nuclear factor kappa B (NFB; Collins et al., 1995; Stancovski and Baltimore, 1997; Winyard and Blake, 1997; Mercurio and Manning, 1999; Bonizzi et al., 2000). In quiescent cells, NF
B (p50/p65 heterodimer) is localized to the cytoplasm by virtue of its association with inhibitory protein kappa B (I
B). I
B apparently masks the nuclear localization sequence on NF
B, and thereby prevents its translocation to the nucleus (Henkel et al., 1992; Lin et al., 1995; Mercurio and Manning, 1999). In cytokine-activated cells, I
B is phosphorylated, ubiquitinated, and subsequently degraded by the proteasome pathway. The loss of I
B allows NF
B to enter the nucleus and initiate the transcription of relevant proinflammatory genes, including those encoding for endothelial adhesion molecules and various cytokines. Interestingly, NF
B transcribes genes encoding for the same cytokines that mobilized it to the nucleus. This positive feedback mechanism could amplify the inflammatory state with severe consequences to the host.
Fortunately, there are negative feedback mechanisms in place that limit an excessive and prolonged inflammatory response on NFB translocation to the nucleus. For example, NF
B transactivates the gene encoding for I
B (Brown et al., 1993; Baldwin, 1996; Bonizzi et al., 2000). The resultant generation of I
B presumably binds to cytoplasmic NF
B and prevents further translocation of this transcription factor to the nucleus. This feedback inhibition assures a transient response to the initiating signal and prevents an excessive, uncontrolled inflammatory response. Our previous preliminary works (Cepinskas et al. 1998. FASEB J. 12(5):A801. Abstract) indicated that there might be an additional negative feedback mechanism in place to control the systemic inflammatory response. In that paper, we noted that the increase in rat myocardial and lung nuclear NF
B induced by sepsis (peritonitis) was enhanced when PMN emigration was prevented by antibodies directed to CD18. This observation suggested that PMN emigration into the lungs and heart during the systemic inflammatory response could reduce tissue nuclear NF
B. Herein, we provide evidence that the IL-1ßinduced increase in endothelial cell monolayer nuclear NF
B can be reduced if PMN are allowed to migrate across these monolayers. Furthermore, engagement of platelet-endothelial cell adhesion molecule 1 (PECAM-1) on endothelial cells by PMN may be the mechanism by which this negative feedback inhibition occurs.
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Results |
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Migrating PMN decrease nuclear NFB in IL-1ß/PAF-stimulated HUVECs
We used confocal microscopy to assess the negative impact of PMN transendothelial migration on the IL-1ßinduced increase in human umbilical vein endothelial cell (HUVEC) nuclear NFB (Fig. 2 A). Under control (unstimulated) conditions, p65 is localized to the cytoplasm of HUVECs (Fig. 2 A, a). After stimulation with IL-1ß/PAF, the p65 is primarily localized to the nuclei of HUVECs (Fig. 2 A, b). When PMN were allowed to migrate across the HUVEC monolayers, there was a decrease in nuclear p65 (Fig. 2 A, c). Of interest is the observation that, although there is some staining for NF
B in the cytoplasm, the overall extent is much less than under control conditions (Fig. 2 A, compare a with c). The reason for the overall lack of cytoplasmic staining after PMN migration is not clear, but may reflect degradation of NF
B or modification of NF
B, such that it is no longer recognized by the antibody.
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Having demonstrated the negative impact of PMN transendothelial migration on the IL-1ßstimulated increase in HUVEC NFB using two different approaches, we assessed whether this phenomenon could be demonstrated using other cytokines to stimulate HUVECs. HUVECs were activated with 10 ng/ml TNF-
(R&D Systems) and 0.5 µg/ml lipopolysaccharide (LPS; Sigma-Aldrich) rather than IL-1ß. Similar results were noted as with IL-1ß, i.e., these compounds increased HUVEC nuclear NF
B, and subsequent PMN transendothelial migration resulted in a decrease in HUVEC nuclear NF
B (unpublished data). These latter observations indicate that PMN transendothelial migration can provide a negative influence on HUVEC nuclear NF
B induced by a variety of cytokines. Because LPS, IL-1ß, and TNF-
have all been implicated in the systemic inflammatory response (Lush and Kvietys, 2000), this negative feedback on HUVEC nuclear NF
B may be very relevant to this pathology. For the remainder of the experiments, we focused on IL-1ß as the cytokine prototype.
PMN-derived soluble factors do not play a role in the decrease in HUVEC nuclear NFB induced by PMN transendothelial migration
In these experiments, we assessed whether PMN secrete or discharge substances that are responsible for the decrease in nuclear NFB of IL-1ßstimulated HUVECs. PMN were separated from HUVEC monolayers by placing them in the apical compartment of cell culture inserts (0.4-µm pore diameter) over the HUVEC monolayers in the basal compartment (distance between PMN and HUVECs was 0.9 mm). When PAF-activated PMN were coincubated with HUVECs in this system for 1 h, there was no detectable increase in nuclear NF
B in naive HUVECs (unpublished data). More importantly, the IL-1ßinduced increase in nuclear NF
B was not affected by subsequent addition of PMN and PAF (unpublished data). These observations suggest that PAF-activated PMN do not release substances that can traverse 0.9 mm to influence HUVEC nuclear NF
B. However, this does not preclude the possibility that contact of PMN with the endothelium is necessary for PMN-derived soluble factors to be effective. Others have shown that adhesion of PMN to biological surfaces renders them more sensitive and reactive to inflammatory stimuli (Fuortes et al., 1993; Furuno et al., 1997).
Previously, we have shown that activated PMN mobilize elastase to the cell surface, where it plays an important role in PMN transendothelial migration (Cepinskas et al., 1999a). Others have shown that neutrophil-derived elastase can also induce cell signaling in epithelial (Hashimoto et al., 1999) and endothelial (Yamaguchi et al., 1998) cells. Thus, several approaches were used to determine whether PMN-derived elastase can decrease nuclear NFB in IL-ß/PAF-stimulated HUVECs. Neutroplasts (neutrophilic cytoplasts) prepared from 10-7 M PAF-stimulated PMN were used, rather that intact PMN. As shown in Fig. 3 A, migrating neutroplasts were also capable of inducing a decrease in HUVEC nuclear NF
B. This observation indicates that PMN degranulation (extracellular release of enzymes) is not required for the negative impact of PMN migration on HUVEC nuclear NF
B. However, the cell membranes of neutroplasts obtained from PAF-activated PMN are enriched in elastase (Cepinskas et al., 1999a). Thus, it was quite possible that membrane-bound elastase could be involved. However, the elastase inhibitor L658 758 (100 µM; Merck) did not affect the decrease in NF
B in HUVEC nuclei induced by migrating intact PMN (Fig. 3 B). In another series of experiments, purified human elastase (0.10.5 µg/ml for 15 min; DakoCytomation) was added to the IL-ß/PAF-stimulated HUVECs. This maneuver also failed to affect the increase in HUVEC nuclear NF
B induced by IL-1ß (unpublished data). Together, these experiments indicate that PMN-derived elastase does not play a role in the negative effect on endothelial nuclear NF
B induced by migrating PMN.
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Adhesive interactions with HUVECs play an important role in the decrease in HUVEC nuclear NFB during PMN transendothelial migration
PMN adhesive interactions with endothelial cells mediated by CD18ICAM-1 are a prerequisite for PMN transendothelial migration (Granger and Kubes, 1994; Kvietys et al., 1996; Panes and Granger, 1998; Wang and Springer, 1998). Thus, we assessed whether these adhesive interactions play a role in the decrease in HUVEC nuclear NFB during PMN migration across HUVEC monolayers. To this end, we assessed the effects of an mAb directed to CD18 (IB4; 40 µg/ml) on the PMN-mediated decrease in nuclear NF
B in IL-ß/PAF-stimulated HUVECs. As shown in Fig. 5 A, inclusion of the mAb prevented the decrease in HUVEC nuclear NF
B; the NF
B level was the same as in the absence of PMNendothelial cell interactions. Based on previous works (Yoshida et al., 1992) and the present paper (legend to Fig. 5), the mAb directed to CD18 decreases adhesion of PMN to HUVECs and prevents the subsequent transendothelial migration (legend to Fig. 5). Although this approach provided useful information, it did not address the issue of whether PMN adhesion to HUVECs or PMN transendothelial migration, per se, was the critical event involved in the decrease in HUVEC nuclear NF
B. Thus, we next assessed the effects of fixed PMN in this system. Fixed PMN adhere to HUVECs, but do not migrate across the monolayers (Cepinskas et al. 1999. FASEB J. 13(4):A178, Abstract and legend to Fig. 5). Fixed PMN failed to induce a decrease in nuclear levels of NF
B in IL-ß/PAF-stimulated HUVECs (Fig. 5 B). Together, these findings suggest that the process of PMN transendothelial migration may be more important than the initial PMN adhesive interactions with HUVECs.
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The effects of PAF-induced PMN transendothelial migration across IL-1ßstimulated HUVECs on the nuclear levels of NFB after a subsequent stimulation of HUVECs with IL-1ß are shown in Fig. 7 A. Lanes 13 of Fig. 7 A show that IL-1ß can induce an increase in HUVEC nuclear NF
B that is dramatically decreased if PMN are allowed to migrate across the HUVEC monolayers. If PMN were not interacted with the HUVEC monolayers, a second challenge with IL-1ß resulted in a greater increase in nuclear accumulation of NF
B than that noted with the first challenge (Fig. 7 A, lane 4). If PMN were allowed to migrate across IL-1ßstimulated HUVEC monolayers, the second challenge with IL-1ß did not result in an increase in HUVEC nuclear NF
B. These findings indicate that allowing PMN to migrate across HUVEC monolayers initially challenged with IL-1ß renders them refractory to a second challenge in terms of NF
B mobilization to the HUVEC nucleus.
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The effects of PAF-induced PMN transendothelial migration across IL-1ßstimulated HUVECs on the PMN transendothelial migration after a subsequent challenge of HUVECs with IL-1ß are shown in Fig. 7 C. The initial stimulation with IL-1ß resulted in an increase in PMN transendothelial migration in response to a chemotactic gradient induced by PAF. If PMN were not allowed to interact with the HUVEC monolayers after the initial challenge with IL-1ß, PAF-induced PMN migration after the second challenge with IL-1ß was similar to that noted after the first IL-1ß challenge. If the PMN were allowed to migrate across the HUVEC monolayers after the initial challenge with IL-1ß, the PAF-induced PMN transendothelial migration after the second IL-1ß challenge was reduced by 45% (Fig. 7 C, compare bar 2 with bar 4).
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Discussion |
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During the course of our experiments, we were aware of one potential caveat to the interpretation of the data. Previous works have shown that, during preparation of samples of endothelial cell monolayers for assay, any PMN associated with the monolayers could lead to artificial proteolysis of proteins under investigation (Moll et al., 1998). In our experiments, we minimized the potential for this artifact to impact on our results in two ways. For EMSA, we allowed the PMN to completely leave the apical aspect of the HUVEC monolayers, and we maximized the antiproteolytic activity of the buffers used to extract nuclear proteins (McDonald et al., 1997). In addition, we used another approach to assess the negative impact of PMN transendothelial migration on the IL-1ßinduced increase in HUVEC nuclear NFB, i.e., confocal microscopy (Fig. 2). This approach involved the fixation of all cells under study, and thus prevented PMN degranulation during sample preparation. Finally, addition of the PMN protease (elastase) directly to IL-1ßstimulated HUVECs had no impact on nuclear NF
B. Together, these observations indicate that artifactual proteolysis was not an issue in our experiments.
PMN adhesion to HUVECs and transendothelial migration is dependent on the interaction of adhesion molecules on both PMN and HUVECs. The firm adhesion of PMN to HUVECs is mediated by CD18ICAM-1 (Granger and Kubes, 1994; Kvietys et al., 1996; Panes and Granger, 1998; Wang and Springer, 1998), whereas transendothelial migration is dependent on homotypic adhesive interactions between PECAM-1 on PMN and HUVECs (Muller, 1995; Liao et al., 1997; Thompson et al., 2001). Furthermore, both endothelial ICAM-I and PECAM-1 can induce intracellular signaling. Cross-linking of ICAM-1 on rat brain endothelial cells promotes intracellular calcium signaling that results in cytoskeletal rearrangement and facilitates lymphocyte transendothelial migration (Etienne-Manneville et al., 2000). Interestingly, recent works indicate that ICAM-1 transfected into CHO cells can support PMN migration across these transfected CHO monolayers; an effect requiring the presence of the cytoplasmic domain of ICAM-1 (Sans et al., 2001). This latter observation indicates that ICAM-1 can contribute to PMN transendothelial migration. Finally, engagement of PECAM-1 on HUVECs with antibodies results in intracellular signaling (increased intracellular calcium); an effect that requires the presence of the intracellular domain (Gurubhagavatula et al., 1998; O'Brien et al., 2001). Thus, we assessed whether cross-linking of either ICAM-1 or PECAM-1 (in the absence of PMN) would have an impact on the levels of nuclear NFB in IL-1ßstimulated HUVECs. Of these two endothelial cell adhesion molecules implicated in PMN transendothelial migration, only engagement and cross-linking of PECAM-1 was capable of mimicking the effects of PMN transendothelial migration on nuclear NF
B in cytokine-stimulated HUVECs (Fig. 6 A). Furthermore, the IL-1ßinduced increase in nuclear NF
B was unaffected by PMN adhesive interactions with endothelial cells derived from PECAM-1deficient mice (Fig. 6 B). A role for PECAM-1 (but not ICAM-1) in intracellular signaling has also been shown in another system (Ferrero et al., 2003). The specific intracellular signals that are initiated by engagement of endothelial cell PECAM-1 by PMN to decrease nuclear NF
B are, at present, unclear and warrant further attention.
Our findings also indicate that the decrease in nuclear NFB in IL-1ßstimulated HUVECs by migrating PMN has functional consequences relevant to the inflammatory process. When PMN transendothelial migration was allowed to occur across IL-1ßstimulated HUVECs, a subsequent challenge with IL-1ß resulted in less (1) nuclear NF
B accumulation; (2) ICAM-1 surface levels; and (3) PMN transendothelial migration (Fig. 7). It is worth noting that although there is a striking loss of NF
B under these circumstances, there are only modest decreases in surface levels of ICAM-1 (48%) and PMN transendothelial migration (45%). These observations indicate that other factors besides NF
B maybe involved in the residual proinflammatory response observed after the second challenge with the cytokine. There may be nuclear transcription factors that are not negatively impacted by PMN migration which may contribute to the inflammatory response. Alternatively, the residual response may be independent of nuclear transcription factors, i.e., other as yet unidentified proinflammatory mediators may be involved. Irrespective of the explanation, these observations indicate that allowing PMN to migrate across IL-1ßstimulated HUVECs results in a decreased proinflammatory response to a second IL-1ß challenge.
The phenomenon described herein is very reminiscent of the development of tolerance to cytokine or LPS stimulation in a variety of cells (Fraker et al., 1988; Laegreid et al., 1995; Lush et al., 2000). However, there is a major difference in the development of this refractoriness to cytokine stimulation between classical tolerance and the tolerance described in the present paper. In classical tolerance, an initial challenge with a cytokine or oxidant stress results in a decreased responsiveness to a subsequent challenge with the same stimulus (Cepinskas et al., 1999b; Lush et al., 2000); an effect independent of PMN interactions with the cells. In the present paper, we have provided evidence that allowing for PMN transendothelial migration between the two challenges can also result in the development of tolerance. To our knowledge, this is the first direct demonstration of such a phenomenon. Further studies are warranted to unravel the mechanisms involved in the development of this form of tolerance.
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Materials and methods |
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Endothelial cells
HUVECs were harvested from umbilical cords by collagenase treatment and cultured as described previously (Yoshida et al., 1992; Cepinskas et al., 1999a). Primary through second passage HUVECs were used in experiments. When necessary, mouse (C57BL/6 wild type or PECAM-1 deficient on C57BL/6 background; The Jackson Laboratory and gift from Dr. W. Muller) myocardial endothelial cells were harvested as described previously (Rui et al., 2001).
Leukocytes
Human neutrophilic PMN were isolated from venous blood of healthy adults using standard dextran sedimentation and gradient separation on HISTOPAQUE®-1077 (Sigma-Aldrich; Cepinskas et al., 1999b). This procedure yields a PMN population that is 9598% viable (trypan blue exclusion) and 98% pure (acetic acid-crystal violet staining). When necessary, PMN cytoplasts (neutroplasts) were prepared as described previously (Cepinskas et al., 1999a). In brief, PMN in 12.5% Ficoll solution were layered over a discontinuous gradient of 16 and 25% Ficoll and centrifuged at 81,000 g. Neutroplasts were harvested from the 12.5%/16% interface. This procedure yields a population of anucleated PMN devoid of granules. For some experiments, PMN were fixed for 5 min using 3% PFA in PBS. When necessary, PMN were isolated from whole blood of mice (C57BL/6 wild type or iNOS-deficient on C57BL/6 background; The Jackson Laboratory) by centrifugation on a NIM-2 gradient (Cardinal Associates, Inc.).
PMN transendothelial migration
PMN transendothelial migration was assessed as described previously (Cepinskas et al., 1997, 1999a). Endothelial cells were grown to confluence on 25 µg/ml fibronectin-coated Falcon cell culture inserts (3-µm-diam pores) and stimulated with 1 ng/ml IL-1ß (R&D Systems) for 4 h. Neutrophils were added to the endothelial cell monolayers and allowed to migrate across them for 1 h in the presence of PAF (10-10 M; Sigma-Aldrich) in the basal compartment. This period of time was sufficient to ensure that the bulk of the PMN had traversed the monolayer. To quantitate changes in the rate of PMN transendothelial migration, PMN were labeled with 51Cr, and PMN migration was assessed 30 min after coincubation with HUVEC monolayers, i.e., before PMN transendothelial migration was complete.
EMSA for NFB
Nuclear protein from whole tissue or endothelial cells was extracted as described previously (McDonald et al., 1997; Cepinskas et al., 1999b). For EMSA, 3 µg of total nuclear proteins was incubated with 1.0 pmol of double-stranded [32P]ATP end-labeled oligonucleotides containing consensus binding sequences for NF-
B (sense strand 5'-AGGGACTTTCCGCTGGGGACTTTCC-3') in a binding buffer (10 mM Hepes, pH 7.9, 80 mM NaCl, 3 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 10% glycerol) as described previously (Bielinska et al., 1990; Cepinskas et al., 1999b). After electrophoresis under nondenaturing conditions (0.5x TBE buffer), the gels were dried and the radioactive bands were visualized on X-ray films.
Laser scanning confocal microscopy for NFB
HUVECs were prepared for immunofluorescence analysis as described previously (Cepinskas et al., 1999a). HUVECs were treated with 2 µg/ml rabbit pAb NFB p65 (A; Santa Cruz Biotechnology, Inc.) and Texas redconjugated secondary goat antirabbit IgG (Molecular Probes, Inc.). The nuclei were stained with Hoechst 33342. The distribution of HUVEC NF
B p65 subunits was analyzed by confocal microscopy.
Cell ELISA for ICAM-1
Cell surface levels of ICAM-1 were measured as described previously (Lush et al., 2000). In brief, PFA-fixed (3%) HUVECs were incubated with primary mAb directed against ICAM-1 (RR1/1, IgG1; Biosource International). The antibody binding intensity was evaluated using a Mouse Extravidin Peroxidase Staining Kit (Sigma-Aldrich).
Adhesion molecule cross-linking
Cross-linking of ICAM-1 was induced as described previously (Etienne-Manneville et al., 2000) with some modifications. In brief, HUVECs were grown in 35-mm Petri dishes and stimulated with 1 ng/ml IL-1ß for 4 h. HUVEC ICAM-1 was engaged by treatment of HUVECs with a primary mAb (20 µg/ml) directed against ICAM-1 (RR1/1, IgG1; Biosource International) for 30 min, and cross-linking was induced by subsequent addition of 5 µg/ml rabbit antimouse antibodies (DakoCytomation) for an additional 30 min. Cross-linking of PECAM-1 was done in a similar manner (Berman and Muller, 1995; Gurubhagavatula et al., 1998) using an mAb (20 µg/ml) against PECAM-1 (hec7, IgG2a; a gift from Dr. W.A. Muller, Cornell University School of Medical Sciences, New York, NY).
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Acknowledgments |
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This work was supported by the Canadian Institutes of Health Research grants MOP 44010 (to G. Cepinskas), and MOP 37758 and 13668 (to P.R. Kvietys).
Submitted: 5 December 2002
Revised: 21 March 2003
Accepted: 25 March 2003
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