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Original Research

Cyclophilin A cooperates with MIP-2 to augment neutrophil migration

Shannon J Heine1 Department of Microbiology, Immunology and Tropical Medicine, The George Washington University, Washington, DC, USA

,

Denise Olive1 Department of Microbiology, Immunology and Tropical Medicine, The George Washington University, Washington, DC, USA

,

Ji-Liang Gao2 Laboratory of Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

,

Philip M Murphy2 Laboratory of Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

,

Michael I Bukrinsky1 Department of Microbiology, Immunology and Tropical Medicine, The George Washington University, Washington, DC, USA

&

Stephanie L Constant1 Department of Microbiology, Immunology and Tropical Medicine, The George Washington University, Washington, DC, USACorrespondence[email protected]

Figures & data

Figure 1 CypA and MIP-2 cooperate in vitro. In vitro chemotaxis assays were set up using purified mouse neutrophils incubated in the presence of a single dose of fMLP (positive control), a single dose of CypA, MIP-2, or KC, or a fixed dose of MIP-2 or KC plus varying doses of CypA. 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, CypA alone, MIP-2 alone, or combinations of MIP-2 plus CypA. B) Mean + SE chemotactic index for neutrophils incubated with fMLP, CypA alone, KC alone, or combinations of KC plus CypA.

Notes: Statistical significance was determined by Student’s t-test, with n = 6 wells per group. *P < 0.05 and ***P < 0.001. These data are representative of >3 independent experiments.

Abbreviations: See list of abbreviations.

Figure 1 CypA and MIP-2 cooperate in vitro. In vitro chemotaxis assays were set up using purified mouse neutrophils incubated in the presence of a single dose of fMLP (positive control), a single dose of CypA, MIP-2, or KC, or a fixed dose of MIP-2 or KC plus varying doses of CypA. 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, CypA alone, MIP-2 alone, or combinations of MIP-2 plus CypA. B) Mean + SE chemotactic index for neutrophils incubated with fMLP, CypA alone, KC alone, or combinations of KC plus CypA.Notes: Statistical significance was determined by Student’s t-test, with n = 6 wells per group. *P < 0.05 and ***P < 0.001. These data are representative of >3 independent experiments.Abbreviations: See list of abbreviations.

Figure 2 CypA and MIP-2 must be present concurrently for in vitro cooperation to occur. In vitro chemotaxis assays were set up using purified mouse neutrophils preincubated in the presence of chemotaxis medium, medium containing 5 or 50 ng/mL CypA, or medium containing 10 or 100 ng/mL MIP-2, for 30 minutes. The cells were then washed and set up in Boyden chambers with medium alone, 5 ng/mL CypA, or 10 ng/mL MIP-2. Cells preincubated in medium alone were set up with a single dose of MIP-2, CypA, or a combination of both as a positive control for cooperative interaction. A chemotactic index was calculated for each group by dividing the number of migrated cells in test wells by the number of cells preincubated in medium that migrated to medium alone. A) Mean + SE chemotactic index of migration to indicated chemoattractants for neutrophils preincubated with medium alone, 5 or 50 ng/mL of CypA. B) Mean + SE chemotactic index of migration to indicated chemoattractants for neutrophils preincubated with medium alone, 10 or 100 ng/mL doses of MIP-2.

Notes: Statistical significance was determined by Student’s t-test, with n = 6 wells per group. *P < 0.05. These data are representative of >3 independent experiments.

Abbreviations: See list of abbreviations.

Figure 2 CypA and MIP-2 must be present concurrently for in vitro cooperation to occur. In vitro chemotaxis assays were set up using purified mouse neutrophils preincubated in the presence of chemotaxis medium, medium containing 5 or 50 ng/mL CypA, or medium containing 10 or 100 ng/mL MIP-2, for 30 minutes. The cells were then washed and set up in Boyden chambers with medium alone, 5 ng/mL CypA, or 10 ng/mL MIP-2. Cells preincubated in medium alone were set up with a single dose of MIP-2, CypA, or a combination of both as a positive control for cooperative interaction. A chemotactic index was calculated for each group by dividing the number of migrated cells in test wells by the number of cells preincubated in medium that migrated to medium alone. A) Mean + SE chemotactic index of migration to indicated chemoattractants for neutrophils preincubated with medium alone, 5 or 50 ng/mL of CypA. B) Mean + SE chemotactic index of migration to indicated chemoattractants for neutrophils preincubated with medium alone, 10 or 100 ng/mL doses of MIP-2.Notes: Statistical significance was determined by Student’s t-test, with n = 6 wells per group. *P < 0.05. These data are representative of >3 independent experiments.Abbreviations: See list of abbreviations.

Figure 3 CXCR2 internalization increases when CypA and MIP-2 are combined. Purified neutrophils were incubated in medium alone or with the indicated combination of 5 ng/mL CypA, 10 ng/mL MIP-2, or 20 ng/mL KC, for 5 minutes. Cells were then washed in PBS and stained with either FITC-conjugated anti-CD147 or FITC-conjugated isotype control Ab, or PE-conjugated anti-CXCR2 or PE-conjugated isotype control AB. A) CD147 expression on cells incubated with CypA alone, MIP-2 alone, or a combination of CypA + MIP-2. B) CD147 expression on neutrophils incubated with CypA alone, KC alone, or a combination of CypA + KC. C) CXCR2 expression on neutrophils incubated with CypA alone, MIP-2 alone, or a combination of CypA + MIP-2. D) CXCR2 expression on neutrophils incubated with CypA alone, KC alone, or a combination of CypA + KC. MFI were calculated and are shown for each group in panel legends. Bar graphs showing average decrease in MFI relative to medium for E) CD147 expression and F) CXCR2 expression were calculated using 3 independent sets of data.

Abbreviations: See list of abbreviations.

Figure 3 CXCR2 internalization increases when CypA and MIP-2 are combined. Purified neutrophils were incubated in medium alone or with the indicated combination of 5 ng/mL CypA, 10 ng/mL MIP-2, or 20 ng/mL KC, for 5 minutes. Cells were then washed in PBS and stained with either FITC-conjugated anti-CD147 or FITC-conjugated isotype control Ab, or PE-conjugated anti-CXCR2 or PE-conjugated isotype control AB. A) CD147 expression on cells incubated with CypA alone, MIP-2 alone, or a combination of CypA + MIP-2. B) CD147 expression on neutrophils incubated with CypA alone, KC alone, or a combination of CypA + KC. C) CXCR2 expression on neutrophils incubated with CypA alone, MIP-2 alone, or a combination of CypA + MIP-2. D) CXCR2 expression on neutrophils incubated with CypA alone, KC alone, or a combination of CypA + KC. MFI were calculated and are shown for each group in panel legends. Bar graphs showing average decrease in MFI relative to medium for E) CD147 expression and F) CXCR2 expression were calculated using 3 independent sets of data.Abbreviations: See list of abbreviations.

Figure 4 The combination of CypA and MIP-2 increases neutrophil calcium flux. Neutrophils (10⁶) were incubated for 30 minutes at 37°C in fluorescent dye followed by exposure to chemoattractants (5 ng/mL CypA alone, 10 ng/mL MIP-2 alone, or CypA and MIP-2 combined) at the time indicated by arrows. Changes in intracellular calcium levels were monitored over time by changes in fluorescence intensity using the FlexStation system (Molecular Devices). The % increase in Ca²⁺ was calculated as the peak fluorescence with the addition of chemoattractants over the average baseline fluorescence of the cells. As a positive control for the ability of cells to flux calcium, dATP was added at 120 seconds, with the following increases in Ca²⁺ over baseline (CypA = 20%, MIP-2 = 22% and CypA + MIP-2 = 20%). These data are representative of 3 independent experiments.

Abbreviations: See list of abbreviations.

Figure 4 The combination of CypA and MIP-2 increases neutrophil calcium flux. Neutrophils (106) were incubated for 30 minutes at 37°C in fluorescent dye followed by exposure to chemoattractants (5 ng/mL CypA alone, 10 ng/mL MIP-2 alone, or CypA and MIP-2 combined) at the time indicated by arrows. Changes in intracellular calcium levels were monitored over time by changes in fluorescence intensity using the FlexStation system (Molecular Devices). The % increase in Ca2+ was calculated as the peak fluorescence with the addition of chemoattractants over the average baseline fluorescence of the cells. As a positive control for the ability of cells to flux calcium, dATP was added at 120 seconds, with the following increases in Ca2+ over baseline (CypA = 20%, MIP-2 = 22% and CypA + MIP-2 = 20%). These data are representative of 3 independent experiments.Abbreviations: See list of abbreviations.

Figure 5 Actin polymerization increases when CypA and MIP-2 are combined. Neutrophils were serum starved in RMPI for 30 minutes at room temperature before BSA Fraction V was added. Cells were then incubated for 5 minutes at 37°C in: A) medium alone, 5 ng/mL CypA alone, 10 ng/ml MIP-2 alone, or a combination of CypA + MIP-2, or B) 20 ng/mL KC alone, 5 ng/mL CypA alone, or a combination of CypA + KC. The cells were immediately fixed in formalin followed by permeabilization in Triton X-100 and staining with FITC-conjugated phalloidin. MFI were calculated and are shown for each group in panel legends. C) Bar graph showing average increases in phalloidin expression relative to medium that were calculated using 2 independent sets of data.

Abbreviations: See list of abbreviations.

Figure 5 Actin polymerization increases when CypA and MIP-2 are combined. Neutrophils were serum starved in RMPI for 30 minutes at room temperature before BSA Fraction V was added. Cells were then incubated for 5 minutes at 37°C in: A) medium alone, 5 ng/mL CypA alone, 10 ng/ml MIP-2 alone, or a combination of CypA + MIP-2, or B) 20 ng/mL KC alone, 5 ng/mL CypA alone, or a combination of CypA + KC. The cells were immediately fixed in formalin followed by permeabilization in Triton X-100 and staining with FITC-conjugated phalloidin. MFI were calculated and are shown for each group in panel legends. C) Bar graph showing average increases in phalloidin expression relative to medium that were calculated using 2 independent sets of data.Abbreviations: See list of abbreviations.

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.

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.

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