In NT2.N and NT2.A cells, and their parent cell line NT2, we sought to: (1) quantify ELR chemokines, (2) determine receptor (CXCR-1 and CXCR-2) expression, and (3) measure the function of the chemokines generated from these cells.
NT2 cells were differentiated into NT2.N cells and NT2.A cells with all trans retinoic acid and mitosis inhibitors. Chemokine concentrations in culture supernatants were determined by ELISA. Immunofluorescence was used to detect CXCR-1 and CXCR-2. RT-PCR was used to determine chemokine and chemokine receptor mRNA. Chemotaxis assays were used to assess function.
ELR chemokines were not detected in supernatants of NT2 or NT2.N cells, although mRNA for GRO-γ/CXCL3 was found in both. In contrast, in NT2.A cells, mRNA and protein were present for GCP-2/CXCL6, GRO-α/CXCL1, GRO-γ/CXCL3, and IL-8/CXCL8. CXCR-1 and CXCR-2 were expressed on NT2, NT2.N, and NT2.A cells detected by immunofluorescent staining and RT-PCR. Supernatants of NT2.A cells resulted in neutrophil chemotactic function of 30.5 ± 3.9 % , greater than NT2 cells (12.3 ± 1.6 % , mean ± SEM, P < 0.01).
We speculate that astrocytes are a source of ELR chemokines in the human CNS and that neurons and astrocytes can respond to those chemokines.