Neutrophil migration toward sites of infection is integral in the innate immune response. Chemokines secreted by resident cells in response to inflammatory stimuli establish a gradient that induces neutrophils to extravasate and migrate to infected tissues. Although beneficial for pathogen clearance, the neutrophilia that ensues during inflammatory responses often results in extensive bystander tissue damage. Neutrophil accumulation and activation within tissues contributes significantly to the pathology of both autoimmune and non-autoimmune inflammatory conditions (reviewed in23
) and can also promote the onset of carcinogenic events (reviewed in24
). Therefore, understanding how neutrophil recruitment is regulated, including the factors that contribute to this regulation, has important therapeutic implications for a wide variety of diseases associated with tissue injury.
Many chemokines are secreted during inflammation, suggesting either a high degree of redundancy or an overlap in function. Interestingly, several chemokines have been shown to work in partnership with the potential to amplify inflammatory responses. For example, studies with the CC plasma chemokine, Regakine-1, showed a synergistic enhancement in neutrophil migration when combined with IL-8 (CXCL8), GCP-2 (CXCL6), MCP-3 (CCL7),25
or with NAP-226
in vitro, and when co-administered with GCP-2 in vivo.27
Interestingly, Regakine-1 was also shown to synergize with a chemotactic anaphylatoxin, C5a,26
suggesting that these types of interactions are not restricted to classical chemokines. Other studies have found that IL-8 cooperates with GM-CSF in the female reproductive tract, resulting in enhanced neutrophil chemotaxis. 28
This enhanced recruitment was inhibited by up to 95% when the 2 chemokines were neutralized in combination.28
RANTES (CCL5) and MCP-1 (CCL2) were shown to work together to enhance the migration of PBMCs in vitro18
and the in vivo co-injection of RANTES and IP-10 (CXCL10) was reported to augment T lymphocyte recruitment.29
IP-10 has also been shown to increase the migration of Th1 and Th2 clones when combined with MDC (CCL22) in vitro.22
Extracellular cyclophilins are another family of proteins with chemoattracting properties that are elevated during inflammatory responses. Our laboratory has previously demonstrated that extracellular cyclophilins contribute significantly to neutrophil recruitment in a mouse model of LPS-induced lung inflammation. 6
Inhibiting the function of cyclophilins reduced the influx of neutrophils by 40%–50% in this model.6
Given the many other neutrophil-recruiting chemokines known to be present following LPS challenge,12
we investigated in the current studies whether cyclophilins might have the capacity to synergize with MIP-2 and/or KC, the major neutrophil-attracting chemokines in this disease.
In initial in vitro chemotaxis studies we observed a potent functional interaction between CypA and MIP-2, but not with KC, that resulted in augmented neutrophil migration. Although several different doses of KC were tested and failed to cooperate with CypA in the combination assays (data not shown), we cannot rule out the possibility that KC and CypA might be able to function in either an additive or synergistic partnership at doses not tested here. Nevertheless, the current findings suggest that partnerships between chemoattractants are likely selective. Recent studies suggest that the family to which a chemokine belongs is a critical parameter in determining whether cooperative interactions will occur. Specifically, these studies showed that chemokines belonging to the same family (for example CXC or CC) are less likely to interact due to their competing for related receptors and/or using similar signaling pathways.20
Both MIP-2 and KC signal through the same receptor, CXCR2, suggesting that the interaction with CypA may occur at a step preceding receptor binding. A possible mechanism is that interactions between MIP-2 and CypA enhance MIP-2 binding and/or signaling through CXCR2, possibly via the peptidyl-prolyl isomerization activity of CypA.
The principal binding and signaling receptor for CypA is CD147, a type I integral membrane protein carrying 2 immunoglobulin-like domains.30
Since CD147 bears no structural homology with any of the 4 major families of chemokine receptors (CR, CCR, CXCR, and CX3CR), extracellular cyclophilins may be less restricted in their capacity to interact and synergize with other chemokines. However, it should be noted that the interaction between CypA/CypB and CD147 has also been shown to require the presence of cell surface heparans for optimal induction of cyclophilin binding and signaling.15
Recent studies have reported that syndecan-1 is the principal heparan co-receptor for CypB, which coordinates with CD147 to activate signaling pathways and induce T cell adhesion and migration.32
Interestingly, KC has also been shown to bind syndecan-1.33
The fact that syndecan-1 is implicated in both cyclophilin and KC signaling pathways could provide an alternative explanation for why we did not observe any cooperation between these 2 chemoattractants in the current studies: CypA and KC may compete for syndecan-1 binding, thus reducing their ability to signal through the corresponding receptor and preventing their capacity to interact.
Of particular interest was the observation that combinations of CypA and MIP-2 mediated increases in neutrophil migration that were greater than additive, suggesting a synergistic interaction. The phenomenon of synergy between chemoattractants in vitro is typically observed only within narrow ranges of concentrations that greatly depend on the cell type.34
Our data fit this criterion in that the synergistic effect was seen only at very specific dose combinations. To provide support that the apparent synergistic interaction between CypA and MIP-2 was also associated with altered signaling events, several biochemical parameters were measured. Combinations of CypA and MIP-2 did indeed lead to an augmented internalization of CXCR2, increased and more prolonged calcium mobilization, and enhanced actin polymerization in neutrophils, although these changes were for the most part additive rather than synergistic. Similar discrepancies in functional outcome versus signaling profiles have been reported during chemokine synergy by other laboratories.35
One possibility is that the specific parameters measured in the current studies are not directly associated with the signaling events required to mediate functional synergy between chemokines. However, a more likely explanation is that synergistic outcomes are due to the integration of multiple signaling events. Thus, while changes in one signaling parameter provide evidence for enhanced receptor-mediated events, these events may represent only one arm of an integrated signaling pathway.
Based on our current findings, we propose the following mechanism for the augmented activation of neutrophils by combinations of CypA and MIP-2. When the 2 chemoattractants are present together, CypA may promote greater and/or more efficient binding of MIP-2 to its receptor CXCR2, resulting in increased CXCR2 internalization (). The internalized receptor-chemokine (CXCR2-MIP-2) complexes may then contribute to increased signaling by promoting receptor recycling. Increases in downstream signaling events are evidenced by an augmentation in calcium flux (). The ensuing increase in actin polymerization () results in a more efficient migration by individual cells, or enables a greater total number of cells to migrate, due to previously sub-optimal thresholds of activation becoming optimal. This is supported by in vivo observations that neutrophil numbers are still high as late as 24 hours after LPS delivery, yet inhibiting cyclophilins at this time point (when cyclophilins are at their peak level) reduces neutrophilia by only 40% to 50%.6
Co-inhibiting CypA and MIP-2 (or any additional chemokines found to cooperate with CypA) provides a potential approach whereby late neutrophil recruitment might be further reduced, resulting in a decrease in the amplitude of the response and the collateral tissue damage associated with prolonged neutrophilia. Indeed, such an outcome was reported when the synergizing chemokines, IL-8, and GM-CSF, were co-inhibited,28
providing preliminary support for this type of therapeutic intervention.