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The isoprostane 8-iso-PGF₂ suppresses monocyte adhesion to human microvascular endothelial cells via two independent mechanisms

Anila Kumar^*, Edward Kingdon and Jill Norman^*,1

^* Centre for Nephrology, Division of Medicine, Royal Free and University College Medical School, London, UK; and
Sussex Kidney Unit, Brighton and Sussex University Hospitals Trust, UK

¹Correspondence: Centre for Nephrology, Division of Medicine, Department of Biochemistry and Molecular Biology (2nd floor), Royal Free and University College Medical School, Royal Free Campus, Rowland Hill St., London NW3 2PF, UK. E-mail: j.norman{at}rfc.ucl.ac.uk

SPECIFIC AIMS

It has been suggested that in addition to being markers of oxidative stress, isoprostanes may have pathogenic functions. The most extensively studied compound is 8-iso-PGF₂. The aim of the present study was to investigate the actions of 8-iso-PGF₂ on microvascular endothelial cells, focusing specifically on the adhesion of monocytes to the endothelium, an early event in the inflammatory response.

PRINCIPAL FINDINGS

1. 8-Iso-PGF₂ dose-dependently inhibits monocyte adhesion to human dermal microvascular cells (HMEC)
In confluent, quiescent and proliferating HMEC, 8-iso-PGF₂ (10^–12–10^–5 M) dose-dependently inhibited adhesion of U937 monocytes, with maximal inhibition at 10^–6 M (Fig. 1 ); 1 µM 8-iso-PGF₂ was used to investigate the mechanism by which isoprostane suppresses monocyte adhesion. Inhibition of adhesion was not due to cytotoxicity of 8-iso-PGF₂ to HMEC or monocytes. In contrast to the inhibitory effect of 8-iso-PGF₂ on monocyte adhesion to HMEC, 8-iso-PGF₂ (10^–10–10^–8 M) stimulated adhesion to HUVEC, confirming a previous report and suggesting diverse effects of the isoprostane on endothelial cells from different vascular beds. Suppression of monocyte adhesion required incubation of HMEC with 8-iso-PGF₂ before addition of monocytes, indicating that inhibition is due to isoprostane-induced changes in HMEC. By flow cytometry, 8-iso-PGF₂ had no effect on HMEC expression of VCAM-1 and ICAM-1. Time course studies (0.5–24 h) revealed that 8-iso-PGF₂ (1 µM) caused a time-dependent inhibition of monocyte adhesion to HMEC, first apparent after 1–2 h exposure to the isoprostane and maximal at 4 h (this treatment period was used in subsequent experiments). 8-iso-PGF₂ inhibited monocyte adhesion to rat renal microvascular endothelial cells, indicating a similar response in capillary endothelial cells from different organs (skin and kidney) and different species (human and rat). Adhesion of THP-1 monocytes to HMEC was inhibited by 8-iso-PGF₂, showing that the effect was not unique to U937 cells.

View larger version (22K): [in this window] [in a new window]   Figure 1. 8-iso-PGF2 and 8-iso-PGF2-CM suppress basal and TNF--induced monocyte adhesion. Confluent, quiescent HMEC were treated with 8-iso-PGF2 (1 µM) or with 8-iso-PGF2(1 µM)-CM in the absence or presence of TNF- (100 U/mL) for 4 h before the addition of U937 monocytes; monocyte adhesion was measured colorimetrically by crystal violet staining. Data are mean absorbance at 595 nm ±SD; n= 3 experiments. *P<0.05 vs. 0 8-iso-PGF2.

2. 8-iso-PGF₂ stimulates production of a secondary inhibitor of monocyte adhesion by HMEC
To determine if 8-iso-PGF₂ suppressed monocyte adhesion directly or indirectly, conditioned medium transfer experiments were performed. Conditioned medium (CM) from HMEC exposed to 8-iso-PGF₂ (0–10^–6 M) for 24 h dose-dependently inhibited monocyte adhesion to naive HMEC, with maximal inhibition induced by CM from cells treated with 1 µM 8-iso-PGF₂ (8-iso-PGF₂(1 µM)-CM) (Fig. 1) . The time course of the CM-induced inhibition was delayed vs. the direct effect of 8-iso-PGF₂, becoming apparent only after 3–4 h of treatment, suggesting a distinct inhibitory activity. CM-induced suppression did not require prior incubation of the HMEC in the absence of monocytes. CM had no effect on monocytes alone, suggesting that, like the isoprostane-induced suppression, CM-induced suppression of adhesion is mediated via changes to the endothelial cells.

3. 8-iso-PGF₂ and 8-iso-PGF₂-CM inhibit TNF--induced monocyte adhesion to HMEC
To determine whether 8-iso-PGF₂ can suppress cytokine-induced as well as basal monocyte adhesion, HMEC were treated with TNF- in the presence or absence of 1 µM 8-iso-PGF₂ or 8-iso-PGF₂ (1 µM)-CM and monocyte adhesion was measured. TNF- (100 U/mL) stimulated adhesion of U937 monocytes to HMEC. 8-iso-PGF₂ suppressed TNF--induced adhesion to below control levels, although the level of adhesion to cells treated with a combination of isoprostane plus TNF- remained above that observed in cells treated with isoprostane alone (Fig. 1) . 8-iso-PGF₂(1 µM)-CM blocked TNF--induced monocyte adhesion, reducing adhesion to a level comparable to that induced by CM alone (Fig. 1) .

4. Differential requirement of 8-iso-PGF₂- and 8-iso-PGF₂-CM-induced suppression of monocyte adhesion for new protein synthesis
To determine whether the inhibition of monocyte adhesion is dependent on new protein synthesis, HMEC were treated with 8-iso-PGF₂ or 8-iso-PGF₂(1 µM)-CM in the presence or absence of the protein synthesis inhibitor cycloheximide (Chx; 1 µM). Chx had no effect on the isoprostane-induced inhibition of monocyte adhesion, suggesting this effect is independent of de novo protein synthesis. In contrast, CM-induced suppression of adhesion was abolished by Chx, indicating that inhibition by the secondary mediator is dependent on new protein synthesis, providing further evidence of two distinct mechanisms of inhibition.

5. Role of the thromboxane A2 receptor in 8-iso-PGF₂- and 8-iso-PGF₂-CM-induced suppression of monocyte adhesion
It has been shown that some biological effects of isoprostanes are mediated via the thromboxane A2 receptor (TP). The inhibitory effect of 8-iso-PGF₂ and 8-iso-PGF₂-CM on monocyte adhesion to HMEC was mimicked by U46619, a TP agonist. SQ29548, a TP antagonist, blocked the 8-iso-PGF₂-induced inhibition of monocyte adhesion but had no effect on the CM-induced inhibition, indicating TP-dependent and -independent mechanisms, respectively (Fig. 2 ).

View larger version (33K): [in this window] [in a new window]   Figure 2. 8-iso-PGF2-induced suppression of monocyte adhesion is mediated by the thromboxane receptor whereas 8-iso-PGF2-CM-induced suppression is thromboxane receptor independent. Confluent, quiescent HMEC were treated with agonist U46619 (10 µM), 8-iso-PGF2 (1 µM), or 8-iso-PGF2(1 µM)-CM in the absence and presence of the thromboxane receptor antagonist SQ29548 (10 µM) for 4 h. Cells were washed and monocyte adhesion measured. Data are mean absorbance at 595 nm ±SD; n = 3 experiments. *P<0.05 vs. 0 8-iso-PGF2.

6. Different intracellular signaling pathways are involved in 8-iso-PGF₂- and 8-iso-PGF₂-CM-induced suppression of monocyte adhesion
A variety of signaling pathways has been implicated in mediating isoprostane-induced changes in cell function. 8-iso-PGF₂-induced inhibition of monocyte adhesion was blocked by the p38 inhibitor SB203580 and the JNK pathway inhibitor curcumin but unaffected by the MEK-1 inhibitor PD98059, suggesting that activation of the p38 and JNK pathways, but not MEK-1/ERK, is required for this effect. CM-induced inhibition was completely blocked by SB203580 and unaffected by PD98059, again implicating activation of p38 without involvement of MEK-1/ERK. However, CM-induced suppression of monocyte adhesion was only partially blocked by curcumin, suggesting a less stringent requirement for activation of the JNK pathway.

CONCLUSIONS AND SIGNIFICANCE

Studies of large vessel endothelial cells have revealed a number of proinflammatory effects of the isoprostane 8-iso-PGF₂. Adhesion of monocytes to the endothelium is an early event in the inflammatory response, and it has been reported that 8-iso-PGF₂ stimulates adhesion of U937 to HUVEC. However, the primary site of inflammation is the microvasculature, and we show here that in microvascular endothelial cells, 8-iso-PGF₂ suppresses adhesion of monocytes to HMEC via both direct and indirect mechanisms (Fig. 3 ). That this may reflect differences in response to 8-iso-PGF₂ of endothelial cells from different vascular beds is supported by our experiments confirming that 8-iso-PGF₂-induced up-regulation of adhesion to HUVEC. We have shown that the inhibitory effect is reproducible using microvascular endothelial cells from different species and organs, indicating the response is not unique to a particular endothelial cell line. Similar results were obtained with two different monocyte cell lines.

View larger version (20K): [in this window] [in a new window]   Figure 3. Schematic diagram. Effect of 8-iso-PGF2 (1 µM) or 8-iso-PGF2(1 µM)-CM on adhesion of monocytes to HMEC. iP: 8-iso-PGF2; TP: thromboxane receptor; iP-CM: soluble factor produced in CM by 8-iso-PGF2-treated cells; R?: hypothetical receptor for CM factor.

Monocytes adhere to endothelial cells by engaging cell surface adhesion molecules: VCAM-1, ICAM-1, and E-selectin have been implicated in this process. Although suppression of monocyte adhesion to HMEC was shown to be mediated by isoprostane-induced changes in endothelial cells rather than to effects on the monocytes, the isoprostane had no effect on HMEC surface expression of ICAM-1 or VCAM-1, suggesting that, under basal conditions, suppression of adhesion is not mediated by down-regulation of these adhesion molecules on the endothelial cell surface. The stimulatory effect of 8-iso-PGF₂ on monocyte adhesion to HUVEC is also independent of changes in adhesion molecule expression. Although flow cytometry analysis showed no changes in VCAM-1 or ICAM-1 expression in response to 8-iso-PGF₂, that isoprostane-induced suppression of monocyte adhesion requires incubation of the endothelial cells with 8-iso-PGF₂ in the absence of monocytes implies that the isoprostane interferes with an endothelial receptor for the monocytes. The mechanism underlying 8-iso-PGF₂ suppression of monocyte adhesion to HMEC remains to be elucidated, but we speculate that the isoprostane may induce release or activation of a protease activity, which cleaves part of a monocyte attachment receptor from the surface of the HMEC, thereby preventing adhesion of the monocyte. The rapidity of the response and the lack of any requirement for de novo protein synthesis support such a post-translational mechanism.

Monocyte adhesion to endothelial cells can be stimulated by cytokines such as TNF- or IL-1. 8-Iso-PGF₂ blocked TNF--induced monocyte adhesion to HMEC, suggesting a potent effect able to over-ride the action of inflammatory cytokines.

Besides a direct inhibitory effect of 8-iso-PGF₂ on monocyte adhesion to microvascular endothelial cells, conditioned medium transfer experiments revealed that the isoprostane time-dependently induces a secondary mediator capable of suppressing basal and cytokine-inducible adhesion of monocytes, but by an alternative mechanism to that used by the isoprostane itself. We speculate that this secondary inhibitory activity may be an iP-induced cleavage product that, in turn, binds to a surface receptor to block monocyte adhesion. This blockade is likely to be indirect, as the inhibitory effect of conditioned medium depends on new protein synthesis. The response is delayed compared with that induced by 8-iso-PGF₂, taking 3–4 h to become apparent.

8-iso-PGF₂ exerts many biological effects via the thromboxane A2 receptor (TP), although there is evidence to suggest the existence of an uncharacterized unique isoprostane receptor(s). Leitinger et al. have shown that stimulation of monocyte adhesion to HUVEC occurred via TP. Here we show that 8-iso-PGF₂-induced inhibition of monocyte adhesion to HMEC seems to occur via this receptor, although different downstream signaling pathways may be activated to elicit different responses. The 8-iso-PGF₂-induced increase in monocyte adhesion in HUVEC and suppression in HMEC appear to involve activation of p38 MAPK, but the former involves activation of ERK1/2 while the latter is independent of this pathway. Suppression of monocyte adhesion by 8-iso-PGF₂-CM is independent of TP, demonstrating another difference between the direct and indirect mechanisms of monocyte adhesion inhibition. The conditioned medium effect primarily involves activation of p38, with only partial dependence on JNK.

These data show that the isoprostane 8-iso-PGF₂ can suppress attachment of monocytes to HMECs via two distinct pathways, which suggests an unexpected, potentially anti-inflammatory effect of the isoprostane in the microvasculature. Characterization of the mechanisms and mediators of 8-iso-PGF₂-induced suppression of monocyte adhesion may facilitate development of novel anti-adhesion therapies.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1364fje;

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