Neutrophil-specific chemokines are produced by astrocytic cells but not by neuronal cells.
BACKGROUND
Neutrophils have a central role in the inflammatory conditions of the central nervous system (CNS). ELR chemokines direct neutrophil migration, but the source of chemokines in the CNS is unclear. We quantified chemokine production using cell-line models of astrocytic and neuronal cells, specifically NT2.N cells, a human line with characteristics of immature neurons, and NT2.A cells, a line with characteristics of astrocytes.


OBJECTIVE
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.


METHODS
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.


RESULTS
ELR chemokines were not detected in supernatants of NT2 or NT2.N cells, although mRNA for GRO-gamma/CXCL3 was found in both. In contrast, in NT2.A cells, mRNA and protein were present for GCP-2/CXCL6, GRO-alpha/CXCL1, GRO-gamma/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).


CONCLUSIONS
We speculate that astrocytes are a source of ELR chemokines in the human CNS and that neurons and astrocytes can respond to those chemokines.
Lu
Wenge
W
Department of Pediatrics, Division of Neonatology, University of South Florida College of Medicine/All Childrens Hospital, St. Petersburg, FL 33701, USA.
Maheshwari
Akhil
A
Misiuta
Iwona
I
Fox
Samuel E
SE
Chen
Ning
N
Zigova
Tanja
T
Christensen
Robert D
RD
Calhoun
Darlene A
DA
D001253:Astrocytes / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D045744:Cell Line, Tumor; D018925:Chemokines / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D005452:Fluoresceins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D009474:Neurons / Q000378:metabolism*; D009504:Neutrophils / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis; D023062:Receptors, Interleukin-8A / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D023063:Receptors, Interleukin-8B / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018925:Chemokines; D005452:Fluoresceins; D012333:RNA, Messenger; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B; C085925:calcein AM