Preferential chemotaxis of activated human CD4+ T cells by extracellular cyclophilin A

Abstract

The recruitment and trafficking of leukocytes are essential aspects of the inflammatory process. Although chemokines are thought to be the main regulators of cell trafficking, extracellular cyclophilins have been shown recently to have potent chemoattracting properties for human leukocytes. Cyclophilins are secreted by a variety of cell types and are detected at high levels in tissues with ongoing inflammation. CD147 has been identified as the main signaling receptor for cyclophilin A (CypA) on human leukocytes. It is interesting that the expression of CD147 is elevated on leukocytes from inflamed tissue, suggesting a correlation among the presence of extracellular cyclophilins, CD147 expression, and inflammatory responses. Thus, cyclophilin-CD147 interactions may contribute directly to the recruitment of leukocytes into inflamed tissues. In the current studies, we show that activated human T lymphocytes express elevated levels of CD147, compared with resting T cells and that these activated T cells migrate more readily to CypA than resting cells. Furthermore, we show that unlike resting CD4+ T cells, the cyclophilin-mediated migration of activated T cells does not require interaction with heparan sulfate receptors but instead, is dependent on CD147 interaction alone. Such findings suggest that cyclophilin-CD147 interactions will be most potent when leukocytes are in an activated state, for example, during inflammatory responses. Thus, targeting cyclophilin-CD147 interactions may provide a novel approach for alleviating tissue inflammation.

Fig. 1

CD147 expression is up-regulated on circulating, activated CD4⁺ T cells. PBMCs obtained from human peripheral blood were stained with TC-conjugated anti-CD4 and FITC-conjugated anti-CD147 mAb or isotype control mAb plus PE-conjugated anti-CD25 or anti-HLA-DR. Dot plots show expression of CD147 or isotype versus CD25 expression (A) or HLA-DR (B) after gating on lymphoid CD4⁺ cells. Circled regions denote populations of cells showing CD147^bright expression.

Fig. 2

CD147 expression is up-regulated on in vitro-activated CD4⁺ T cells. PBMCs obtained from human peripheral blood were stimulated in vitro with PHA for 48 h. These activated cells, as well as circulating PBMCs from the same donor, were stained with TC-conjugated anti-CD4 and FITC-conjugated anti-CD147 plus PE-conjugated anti-CD25 or PE-conjugated anti-HLA-DR. Dot plots show expression of CD147 versus CD25 (A) and CD147 versus HLA-DR (B) on circulating and PHA-activated CD4⁺ T cells. Circled regions denote populations of cells showing CD147^bright expression.

Fig. 3

Activated CD4⁺ T cells migrate more readily to CypA than nonactivated cells. The capacity of recombinant CypA to induce migration of nonactivated versus activated CD4⁺ T cells was examined using 48-well Boyden chemotaxis chambers. Populations of activated cells were generated by stimulating human PBMCs with PHA or SEA, followed by MACS separation to isolate CD4⁺ T cells. These activated CD4⁺ T cells were compared with circulating CD4⁺ T cells isolated from the same donor. The migratory response of PHA-activated (A) or SEA-activated (B) CD4⁺ T cells versus nonactivated (circulating) CD4⁺ T cells to 100 ng/ml CypA is shown. (C) Serial dilutions of activated CD4⁺ T cells mixed with nonactivated CD4⁺ T cells were stimulated in the presence of 100 ng/ml CypA. Bar graphs in all panels show mean (±SE) chemotactic index (number of cells migrating in response to chemotactic agent/number of cells migrating to medium alone) for each group, and n = 4 wells per group. Dashed lines delineate significant chemotaxis (>1.2).

Fig. 4

CD147 expression on activated CD4⁺ T cells is comparable with expression on neutrophils and monocytes. PBMCs obtained from human peripheral blood were stimulated in vitro with PHA for 24 h. The next day, a fresh blood draw was used to obtain PBLs, which served as a source of nonactivated T cells, neutrophils, and monocytes. Cells were stained with FITC-conjugated anti-CD147 or isotype control mAb plus TC-conjugated anti-CD4 or APC-conjugated CD14 to gate on neutrophils (CD14-negative) and monocytes (CD14-positive). Histograms show expression of CD147 on individual populations of leukocytes after gating on their representative markers.

Fig. 5

CypA-mediated migration of CD4⁺ T cells is CD147-dependent. (A) Activated and circulating (nonactivated) CD4⁺ T cells were stimulated with 100 ng/ml CypA, with or without 10 μg/ml anti-CD147 or isotype control mAb. (B) Activated CD4⁺ T cells were stimulated with 100 ng/ml CypA or 1 ng/ml RANTES, with or without 10 μg/ml anti-CD147. Graphs show mean (±SE) chemotactic index for each group, and n = 4 wells per group. Dashed lines delineate significant chemotaxis (>1.2). (A and B) Studies were performed using cells from different donors.

Fig. 6

CypA-mediated migration of activated CD4⁺ T cells does not require cell surface heparans. Groups of nonactivated and activated PBMCs were treated with Heparinase I prior to CD4⁺ T cell isolation. Each group was then tested for cyclophilin-mediated chemotaxis using 100 ng/ml CypA, with or without 10 μg/ml anti-CD147 mAb. (A) Bar graphs show the CypA-mediated chemotaxis of untreated versus heparinase-treated, nonactivated and activated CD4⁺ T cells. (B) Bar graphs show the CypA-mediated chemotaxis of activated CD4⁺ T cells and neutrophils, with and without heparinase treatment. (C) Same groups as A, with additional groups of heparinase-treated cells, which included anti-CD147 mAb during chemotaxis. All graphs show mean (±SE) chemotactic index for each groups, and n = 4 – 6 wells per group. Dashed lines delineate significant chemotaxis (>1.2). Studies were performed using cells from different donors.