Heparin-induced thrombocytopenia (HIT), without or with thrombosis, is a serious complication of heparin therapy. Work done in several laboratories including ours has shown that patients with HIT almost invariably have antibodies specific for a platelet derived CXC chemokine, platelet factor 4 (PF4; CXCL4), modified by an external agent, heparin. In the past decade, much has been learned about the pathogenesis of HIT. However, knowledge of the molecular nature of the immune-response is far from complete. We have recently reported that human PF4 modulates, in an opposite manner, CD4+CD25+ regulatory vs. CD4+CD25− non-regulatory T cells and have proposed that a transient impairment of the T regulatory (Tr) cell suppressor activity might be postulated in otherwise healthy individuals, experiencing acute and severe platelet destruction, as it is the case in HIT, systemic lupus erythematosus (SLE), and autoimmune thrombocytopenic purpura (AITP), in which the trigger for the primary immune response is poorly understood. For many years identification of a receptor for the chemokine PF4 eluded discovery. Recently, Lasagni et al. have identified a spliced variant of CXCR3 (CXCR3B) to which PF4 binds with high affinity. While it is not known which CXCR3 isoform is expressed on CD4+CD25+ Tr cells, Lasagni and al. (

J Exp Med.
2003
;
197
:
1537
–49
) reported the intriguing observations that engagement of CXCR3A by their ligands (CXCL9, CXCL10, and CXCL11), is associated with a proliferative response, whereas an opposite effect, e.g. inhibition of cell proliferation, is seen for CXCR3B engagement by the same ligands. In addition, as already noted, PF4, that binds with high affinity to the CXCR3B variant only, has been reported to mediate inhibition of different cell types including hematopoietic progenitors, endothelial cells, and activated T cells. We assessed the relative abundance of CXCR3B (the receptor for PF4) and CXCR3A (not a receptor for PF4) in CD4+CD25+ Tr cells and CD4+CD25− T cells in the presence or the absence of PF4 at different time points. We used a TaqMan quantitative real time PCR (qRTPCR) to determine the mRNA transcription profiles of CXCR3B and A in CD4+CD25+ and CD4+CD25− T cells in the absence or the presence of PF4. mRNA levels were quantitated by comparing experimental levels to standard curves generated using serial dilutions of the same amount of the cloned plasmid DNA for human CXCR3B and A. In CD4+CD25+ Tr cells we found a significant increase of the cDNA copy number encoding CXCR3B (4.67x104 vs. 1.93x104 copies, respectively) in the presence of PF4, while the levels of expression were substantially unchanged for CXCR3A (4.74x104 vs. 4.91x104 copies, respectively). On the contrary, in CD4+CD25− T cells, under the same culture conditions in the presence or the absence of PF4, the expression levels were unchanged for CXCR3B (7.98x104 vs. 7.66x104, respectively) and only slightly increased for CXCR3A (8.77x104 vs. 1.02x105, respectively). Because commercially available antibodies cannot distinguish between the non-spliced (CXCR3A) and the B form of CXCR3, we developed rabbit IgG specific for selected peptides corresponding to the 51 N-terminus AA residues unique to CXCR3B. Using these antibodies we confirmed by flow cytometry that the surface expression of CXCR3B is enhanced on CD4+CD25+ Tr cells primed by PF4. Our data suggest that CXCR3B expression varies differentially according to both cell type and activation status.

Topics:
antigens, cd25, platelet factor 4, t-lymphocytes, antibodies, heparin, ligands, rna, messenger, systemic lupus erythematosus, chemokines, cxc chemokines

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2005, The American Society of Hematology
2004
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