Cyclophilin A cooperates with MIP-2 to augment neutrophil migration

Abstract

Background

Chemokines contribute to inflammatory responses by inducing leukocyte migration and extravasation. In addition, chemoattractants other than classical chemokines can also be present. Many chemokines have been demonstrated to cooperate, leading to an augmentation in leukocyte recruitment and providing a potential role for the presence of multiple chemoattractants. Extracellular cyclophilins are a group of alternative chemotactic factors, which can be highly elevated during various inflammatory responses and, as we have previously shown, can contribute significantly to neutrophil recruitment in an animal model of acute lung inflammation. In the current studies we investigated whether the most abundant extracellular cyclophilin, CypA, has the capacity to function in partnership with 2 classical chemokines known to be secreted in the same model, macrophage inflammatory protein (MIP)-2/CXCL2 and keratinocyte chemoattractant (KC)/CXCL1.

Methods

Neutrophil migration in response to combinations of CypA and MIP-2 or CypA and KC was measured by in vitro chemotaxis assays. Biochemical responses of neutrophils incubated with the combinations of chemoattractants were determined by changes in chemokine receptor internalization and actin polymerization measured by flow cytometry, and changes in intracellular calcium mobilization measured with a calcium sensitive fluorochrome.

Results

A combination of CypA and MIP-2, but not KC, augmented neutrophil migration. Based on the level of augmentation, the cooperation between CypA and MIP-2 appeared to be synergistic. Evidence that CypA and MIP-2 cooperate at the biochemical level was demonstrated by increases in receptor internalization, calcium mobilization, and actin polymerization.

Conclusion

These findings provide evidence for the capacity of extracellular cyclophilins to interact with classical chemokines, resulting in greater and more efficient leukocyte recruitment.

Keywords:

• chemokine
• chemotaxis
• inflammation

Supplementary figure

Figure S1. Dose responses for CypA, MIP-2, and KC-mediated mouse neutrophil chemotaxis. In vitro chemotaxis assays were set up using purified mouse neutrophils incubated in the presence of a single dose of fMLP (positive control) and increasing doses of CypA, MIP-2, and KC. A chemotactic index was calculated for each group by dividing the number of migrated cells in test wells by the number of cells that migrated to medium alone. A) Mean ± SE chemotactic index for neutrophils incubated with fMLP and increasing doses of CypA. B) Mean ± SE chemotactic index for neutrophils incubated with fMLP and increasing doses of MIP-2. C) Mean ± SE chemotactic index for neutrophils incubated with fMLP and increasing doses of KC. N = 6 wells for each group.

Abbreviations: See list of abbreviations.

Acknowledgment

These studies were supported by NIH grant R01AI067254.

Disclosure

The authors report no conflicts of interest in this work.