Endogenous GC and MIF are key molecules in the regulation of the inflammatory response in vivo
. GC have potent anti-inflammatory and immunosuppressive effects including inhibition of pro-inflammatory cytokine production and adhesion molecule expression (12
). In contrast, MIF has a key role in promoting the development of inflammatory diseases via its effects on cytokine and mediator expression (4
). Although its expression can be induced by GC, a key function of MIF is its ability to antagonize the anti-inflammatory effects of GC both in vitro
and in vivo
). More recently, MIF has been shown to promote a hallmark of inflammation, leukocyte recruitment (21
). The ability of MIF to counteract the anti-inflammatory effects of GC raises the possibility that reduced leukocyte recruitment seen in MIF−/−
mice is due to enhanced effects of endogenous GC in the absence of MIF. The findings we report here demonstrate that endogenous MIF and GC have opposing but independent effects on leukocyte trafficking, arguing against an interaction between these molecules in their regulation of inflammatory responses. Furthermore, these findings provide support for the potential anti-inflammatory effectiveness of MIF inhibition, as the reduction in leukocyte-endothelial cell interactions observed in MIF−/−
mice was comparable to that induced by administration of exogenous GC.
Previous in vivo
studies have shown that DEX treatment attenuates LPS-induced leukocyte-endothelial cell interactions (3
). This was supported by the results of the present study, in which pretreatment of wild-type mice with DEX caused reductions in LPS-induced leukocyte rolling flux, adhesion and emigration. However, in MIF−/−
mice, DEX was unable to further reduce leukocyte-endothelial cell interactions across a range of LPS doses. This may indicate that the mechanisms whereby exogenous GC inhibit leukocyte recruitment overlap with those altered by MIF deficiency. These results would argue that MIF inhibition provides a robust anti-inflammatory effect which is comparable to that of GC. However, this finding suggests that under conditions of MIF inhibition, there may be no added benefit of exogenous GC treatment.
Further, these findings could be interpreted to indicate that the independence or otherwise of the actions of MIF and GC depends on the source of the GC. While endogenous GC, as demonstrated by RU486 treatment, and MIF are each able to mediate effects independently of each other, exogenous GC, i.e. DEX, only works effectively in MIF-expressing mice. The reason for this apparent divergence between the effects of endogenous and exogenous GC is unclear. However, it may reflect dose-dependent effects on activation of the GC receptor by GC present at either physiological (endogenous GC) or pharmacological (DEX) levels. Or alternatively, the exogenous version of GC may have a different pharmacokinetic profile in the circulation than that of endogenous corticosterone.
Conversely, GC receptor blockade via RU486 caused significant increases in leukocyte adhesion and emigration in both wild-type and MIF−/−
mice, indicating that the anti-inflammatory effects of endogenous GC are MIF-independent. Moreover, MIF−/−
mice were consistently found to have reduced leukocyte-endothelial cell interactions, regardless of GC receptor blockade, indicating that reductions in leukocyte recruitment in MIF−/−
mice were independent of endogenous GC. One potential explanation for this observation is that MIF and GC regulate recruitment via distinct mechanisms. GC have been shown to inhibit expression of many molecular pathways with roles in leukocyte trafficking, including adhesion molecules and chemokines, as well as leukocyte motility (12
). Expression of the adhesion molecule P-selectin is particularly relevant to the model of inflammation used in this study, as we have observed that this molecule is responsible for greater than 90% of the rolling interactions observed in this model (21
). P-selectin expression has been shown to be inhibited by both endogenous and exogenous GC (32
). In contrast, our previous studies in MIF−/−
mice demonstrated that reductions in leukocyte trafficking in these animals were not associated with any effect on endothelial P-selectin expression or leukocyte P-selectin binding activity (21
). Together, these findings indicate that GC but not MIF have the ability to modulate the P-selectin pathway.
In contrast, the present study provides the first demonstration of the ability of endogenous MIF to regulate endothelial VCAM-1 expression. MIF−/−
mice showed significantly reduced VCAM-1 expression following LPS treatment, proving a plausible mechanism whereby MIF deficiency results in reduced leukocyte recruitment. In contrast, a comparable effect on ICAM-1 expression was not observed. Interestingly, we have previously reported that exposure to exogenous MIF does not increase VCAM-1 expression in the uninflamed microvasculature, under conditions where it is expressed constitutively at functional levels (21
). Taken together with the present findings, these data demonstrate that endogenous MIF plays a role in enabling maximal upregulation of VCAM-1 in response to LPS stimulation, but that exogenous MIF alone is incapable of increasing endothelial VCAM-1 expression.
In regards to the effects of exogenous GC on adhesion molecule expression, DEX has been shown to inhibit ICAM-1 expression in vivo
). Similarly, expression of VCAM-1 in vivo
has been shown to be reduced by exogenous GC, therefore we anticipated increased expression following GC receptor blockade (45
). Unexpectedly, RU486 treatment, while resulting in increased leukocyte-endothelial cell interactions, was associated with reduced VCAM-1 expression in the cremasteric microvasculature in both wild-type and MIF−/−
mice, findings supported by reduced mRNA levels in the gastrocnemius muscle. The explanation for this finding, which suggests that endogenous GC act to promote VCAM-1 expression, is unclear. However it suggests that the effects of endogenous GC do not always mimic the actions of exogenous GC. The alternative explanation, that partial agonist effects of RU486 underlie the unexpected reduction in VCAM-1 expression in RU486-treated mice, is not supported by numerous in vivo
studies in which RU486 has a clear net GC-antagonist effect, resulting in the promotion of inflammation (24
The breadth of the anti-inflammatory effects of GC has been related to GC inhibition of multiple signaling pathways. GC inhibit activation of the NF-κB pathway, and through the induction of MAPK phosphatase-1 (MKP-1), inhibit MAPK activation, with resultant reductions in expression of cytokines, chemokines and adhesion molecules (1
). Both of these signaling pathways are known to promote multiple aspects of leukocyte recruitment (23
). In contrast, the pro-inflammatory effects of MIF are predominantly associated with activation of MAPK family members including ERK and p38 (41
). Correspondingly, cells derived from MIF−/−
mice exhibit decreased MAPK activation (1
). There is minimal evidence, in contrast, that MIF directly activates the NF-κB pathway (30
), but MIF has recently been demonstrated to inhibit GC-induced MKP-1 expression (1
). These divergences between the signaling pathways regulated by MIF and GC may explain the present observations of independent actions of these mediators.
We also investigated the ability of both MIF and endogenous GC to regulate recruitment via an effect on the expression of the chemokines KC, CCL2, MIP-2 and RANTES. Expression of CCL2 and RANTES, but not MIP-2, has been previously reported to be inhibited by exogenous GC, i.e. DEX, in vivo
), whereas results of experiments assessing the sensitivity of KC expression to DEX have been variable (56
). In the present study, the effect of LPS on chemokine expression was comparable both in wildtype and MIF−/−
mice, and in the presence and absence of GC receptor blockade, with the exception of RANTES, which was reduced in MIF−/−
mice. These data suggest that, at least under the conditions used in the present studies, regulation of expression of KC, CCL2 or MIP-2 does not underlie the effects of MIF and endogenous GC on leukocyte recruitment.
Comparison of microvascular responses in wild-type and MIF−/−
mice treated with the higher dose of LPS (100 ng) revealed significantly elevated shear rates in MIF−/−
mice (). It is possible that the anti-adhesive forces provided by the elevated shear rate contributed to the reduced leukocyte-endothelial cell interactions seen in the mutant strain. MIF has been proposed as a critical regulator of vascular tone in septic shock (63
), potentially via its ability to promote production of the vasodilators nitric oxide and PGE2 (27
). Despite this, a comparable significant elevation in shear was not observed at the lower dose of LPS used (10 ng, ) indicating that altered shear alone is insufficient to explain the consistent reduction in leukocyte trafficking observed in MIF−/−
GC remain the mainstay therapeutic modality in some forms of chronic inflammation, particularly, rheumatoid arthritis. While the present studies show clear independence of MIF and endogenous GC during a relatively short term (4–5 hrs) form of inflammation, whether this applies under more extended inflammatory conditions remains unclear. There is evidence that the glucocorticoid receptor, through which GC mediate their effects, is reduced in patients treated with GC, and in animals undergoing a 24 hr sepsis response and GC treatment (28
). These observations would indicate that the milieu of extended inflammation and ongoing exposure to therapeutic levels of exogenous GC has the potential to alter GC-mediated responses, and potentially the interaction with the MIF pathway.
In conclusion, the current study indicates that endogenous MIF and GC have powerful and reciprocal effects on leukocyte trafficking, wherein LPS-induced leukocyte recruitment is suppressed by endogenous GC and promoted by MIF. The demonstration that the effects of endogenous GC are independent of the presence or absence of MIF, and that the effects of endogenous MIF are independent of the effects of endogenous GC, disproves the hypothesis that the effects of MIF are limited to the antagonism of GC. Moreover, the observation that the magnitude of the effects of MIF deficiency was similar to that of exogenous GC administration also argues in favour of the potential effectiveness of anti-MIF therapeutic strategies in inflammatory disease.