In the present study, we provide an initial assessment of the contribution of CXCR4 to the pathophysiology of poly-trauma. Blockade of CXCR4 with AMD3100 resulted in significantly enhanced increases in leukocyte counts in injured animals, when compared with its effects in uninjured animals. Furthermore, AMD3100 increased fluid requirements to maintain hemodynamics and leukocyte reactivity in response to PRR activation after polytrauma. Collectively, these data support the notion that CXCR4 could play an important role in the regulation of the inflammatory response to severe trauma.
The injuries in our model created a pathophysiological condition that closely resembled the typical clinical characteristics of a polytrauma patient. In the absence of hemorrhagic shock, animals demonstrated a 30–40% reduction of MAP during the shock phase, showed an increase in lactate levels and were fluid dependent to maintain hemodynamics. The continuous i.v. fluid requirements demonstrate that the animals were not stabilized within the observation period. As most commonly seen in patients with blunt chest trauma, gas exchange parameters were not affected within the early resuscitation period when ventilated with PEEP and increased FiO2.
AMD3100 is a tight binding, slowly reversible antagonist of CXCR4, which does not induce receptor internalization (33
). However, the affinity of AMD3100 for CXCR4 (Ki
[equilibrium inhibition constant]: 100–650 nmol/L [34
]) is lower than the affinities of SDF-1α (Kd
[equilibrium dissociation constant]: 1–54 nmol/L [34
]) and ubiquitin (Kd
: 100 nmol/L [12
]). The pre-requisite of this study is that the administered dose of AMD3100 prevents lig-and binding to CXCR4 in vivo
, similar to its described effects in cell systems (12
). After i.v. administration, the plasma half-life of AMD3100 is 1–3 h and its volume of distribution is 0.3 L/kg, suggesting distribution beyond the blood compartment (42
). Because quantifications of AMD3100 plasma concentrations or CXCR4 cell surface expression are unable to prove biological activity of AMD3100, we confirmed biological activity through quantification of FITC-ubiquitin receptor binding after i.v. administration. These measurements documented efficacy of i.v. AMD3100 to reduce ubiquitin receptor binding by 60–80%. The observed recovery of ubiquitin receptor binding is consistent with the pharmacokinetic characteristics of AMD3100 (42
). The determined changes in Bmax
after i.v. AMD3100, however, probably underestimate its true effects because AMD3100 will partially dissociate from the receptor during isolation of PBMCs.
Within a few hours after i.v. and subcutaneous injection, AMD3100 induces general leukocytosis and mobilization of CD34+
hematopoietic progenitor cells from the bone marrow (42,52–54). Because AMD3100 administration results in a modest shift to band forms but does not increase metamyelocytes or myelocytes in the blood, the AMD3100- induced leukocytosis was attributed to the demargination of leukocytes from the endothelium (42
). Our findings that AMD3100 mobilized leukocytes into the systemic circulation and the absence of noticeable adverse effects in anesthetized uninjured pigs are consistent with its pharmacological properties that have been described in human volunteers (42
). However, the increase and duration of leukocyte mobilization was slightly reduced in pigs (1.9-fold), as compared with humans (2.5-fold) (42
As described in mice previously, administration of AMD3100 led to a rapid increase of plasma SDF-1α concentrations (55
), suggesting a regulatory feedback loop that controls systemic expression of SDF-1α. The measured plasma concentrations of ubiquitin and the pharmacokinetics of exogenous ubiquitin after i.v. administration are in line with previous findings in pigs (24,25,27,30). The observation that i.v. AMD3100 did not affect ubiquitin plasma levels further supports the assumption that the majority of extracellular ubiquitin originates from its passive release from cells and tissues undergoing physiological turnover or damage (16–18,37,56).
We have shown previously that after i.v. injection, only 10% of exogenous ubiquitin can be recovered in the urine (27
). The biodistribution of exogenous ubiquitin after i.v. injection, however, is currently unknown. Because it has been shown that SDF-1α is predominantly released from bone marrow stromal cells after AMD3100 administration, the finding that coadministration of ubiquitin plus AMD3100 did not influence the AMD3100-induced SDF-1α release in the present study could be explained by insufficient accumulation of ubiquitin in bone marrow (55
). On the other hand, it cannot be excluded that ubiquitin functions as a partial or functionally selective CXCR4 agonist in vivo
, which could correspond to recent findings suggesting that CXCR4 contains separate binding sites for SDF-1α and ubiquitin (13
In contrast to uninjured animals, AMD3100 increased fluid requirements to maintain hemodynamics in animals during resuscitation from polytrauma. Because hematocrit values and hemoglobin concentrations were indistinguishable between the polytrauma groups with and without AMD3100 treatment, these data document that the intravascular fluid status was comparable in all groups and suggest that AMD3100 increased vascular permeability. We have shown previously that treatment with exogenous ubiquitin reduces systemic fluid requirements to maintain hemodynamics and tissue edema formation in various models of inflammation (23–25,27), including the pig polytrauma model that we used in the present study (30
). Along with the finding of the present study that coadministration of AMD3100 and exogenous ubiquitin neutralized the AMD3100-induced increase in fluid requirements, these data imply that CXCR4 activation protects against inflammation-induced capillary leakage.
Moreover, AMD3100-induced effects on leukocyte counts were significantly enhanced after polytrauma, compared with the AMD3100-associated effects in uninjured animals. This result provides initial evidence that CXCR4 functions as an important regulator of leukocyte mobilization and trafficking after severe blunt injuries.
Increased SDF-1α plasma levels were shown to result in elevated blood leukocyte counts (57
). Furthermore, we have shown recently that ubiquitin also exerts chemotactic activity via CXCR4, albeit weaker than SDF-1α (15
). Thus, the increased systemic levels of the CXCR4 agonists likely contribute to the leukocyte-mobilizing effects of AMD3100 and may explain the prolonged elevation in leukocyte counts after AMD3100 and ubiquitin coadministration, possibly through the generation of a reverse chemotactic gradient (57
We could not detect significant differences in lung MPO activities among the groups. Interpretation of this finding, however, is difficult for several reasons. Besides the limitation of a single time point measurement, mechanical ventilation alone is known to result in significant leukocyte infiltration into the lung at a comparable time point (58
). Furthermore, variations of the proportion of blood that is present in lungs will also affect MPO activity. Thus, further studies are required to assess whether the increase in peripheral blood leukocytes by AMD3100 is also accompanied by changes in leukocyte infiltration into tissues after polytrauma.
Moreover, we detected that in vitro AMD3100 treatment increased TNF-α release of normal whole blood upon stimulation with LPS and LTA and that in vivo AMD3100 treatment enhanced TNF-α and IL-6 release of whole blood upon stimulation with LPS or LTA after poly-trauma. The latter could be prevented by coadministration of ubiquitin.
Although we cannot clarify whether leukocytes that were mobilized into the systemic circulation by AMD3100 show enhanced responses upon LPS and LTA stimulation, the detected in vitro
effects of AMD3100 in normal whole blood suggest that endogenous activation of CXCR4 limits proinflammatory responses of the physiological leukocyte population upon activation of the PRRs TLR-2 and TLR-4. This assumption is further supported by previous findings indicating that CXCR4 interacts with TLR-2 and TLR-4 (59
), consistent with the previously described in vitro
and in vivo
effects of exogenous ubiquitin (16
Interestingly, enhanced leukocyte responses were not detectable until the AMD3100-induced increase in SDF-1α plasma levels returned to baseline. Because the CXCR4 agonist CTCE-0214 was recently shown to suppress TNF-α plasma levels in murine endotoxemia and IL-6 production in LPS-stimulated murine bone marrow–derived macrophages (19
), the observation that increased leukocyte responses upon LPS/LTA stimulation were not detectable after AMD3100 administration in uninjured animals and occurred delayed in injured animals could be explained by SDF-1α–mediated activation of CXCR4 on circulating leukocytes. Furthermore, it is conceivable that the AMD3100-induced SDF-1α release counteracted more pronounced physiological consequences of AMD3100. Thus, the present study likely underestimates the pathophysiological role of CXCR4 after tissue injury. CXCR4, SDF-1α and ubiquitin gene knockout, however, results in intra-uterine or perinatal mortality (4
), and reagents that reliably neutralize ubiquitin and SDF-1α are not available. Therefore, in vivo
studies aimed to neutralize SDF-1α or ubiquitin after polytrauma are currently not feasible.
Whereas SDF-1α is a CXCR4 and CXCR7 agonist, ubiquitin is a CXCR4 agonist, but does not bind to CXCR7 (12,13,15,61). Besides being a CXCR4 antagonist, AMD3100 is also a CXCR7 lig-and with weak allosteric agonist activity and increases binding of SDF-1α to CXCR7 (62
). Moreover, AMD3100 may also have partial agonist activity on CXCR4 in certain cell types (63
). Because the observed effects of AMD3100 on fluid requirements and leukocyte function could be neutralized with ubiquitin, these effects are likely attributable to CXCR4. We cannot exclude, however, that CXCR7 activation by SDF-1α and AMD3100, as well as partial CXCR4 agonist activity of AMD3100, may also have contributed to the overall physiological responses that we observed in the present study. Although CXCR4 blockers other than AMD3100 are available, such as IT1t, it remains to be determined whether their actions are strictly confined to neutral CXCR4 antagonism (64