CD40-CD40 ligand interactions in human microglia induce CXCL8 (interleukin-8) secretion by a mechanism dependent on activation of ERK1/2 and nuclear translocation of nuclear factor-kappaB (NFkappaB) and activator protein-1 (AP-1)

Abstract

CXCL8 is a CXC chemokine that recruits leukocytes to sites of inflammation. Expression of CXCL8 in the CNS has been demonstrated in neuroinflammatory diseases, including human immunodeficiency virus (HIV-1) encephalitis, but the mechanism of secretion of this chemokine is not fully understood. CD40 is a 50-kDa protein on the surface of microglia, and we have previously shown that it is increased in expression in HIV-1-infected brain tissue as well as by interferon-gamma (IFNgamma) in tissue culture. We examined the expression and regulation of CXCL8 in cultured human fetal microglia after ligation of CD40 with soluble trimeric CD40 ligand (sCD40L) as well as the expression of CXCL8 on microglia in HIV encephalitic brain tissue sections. Treatment of cultured microglia with IFNgamma + sCD40L resulted in significant induction of CXCL8. This expression was mediated by activation of the ERK1/2 MAPK pathway, as demonstrated by ELISA and Western blot using a specific inhibitor (U0126). Gel shift analyses demonstrated that NFkappaB and AP-1, but not C/EBPbeta, mediate microglial CXCL8 production. We also found increased colocalization of CXCL8 with CD68/CD40-positive cells in HIV encephalitic brain tissue compared with HIV-infected nonencephalitic and normal tissue. Thus, CD40-CD40L interactions facilitate chemokine expression, leading to the influx of inflammatory cells into the CNS. These events can lead to the pathology that is associated with neuroinflammatory diseases.

Fig. 1

IFNγ + sCD40L induces CXCL8 protein and mRNA expression in microglia. A: Microglia were treated with IFNγ alone, sCD40L alone, or IFNγ + sCD40L or were left untreated for 24 hr, after which the supernatants were collected and CXCL8 chemokine ELISAs were performed as described in Materials and Methods. Shown are the means of four separate experiments ± SEM. *P < 0.0009 compared with untreated; **P < 0.004 compared with sCD40L only. B: Microglia were treated with IFNγ alone, sCD40L alone, or both IFNγ + sCD40L or were left untreated for 12 hr. Shown is a representative RPA of two experiments. C: Densitometric analysis comparing CXCL8 to GAPDH expression confirms that IFNγ + sCD40L treatment increases mRNA expression compared with other treatments.

Fig. 2

CXCL8 protein expression is mediated by the ERK1/2 MAPK pathway. CXCL8 protein levels were determined as a percentage difference compared with IFNγ + sCD40L (set to 100%). Microglia were pretreated with 10 μM of the MEK1/2 MAPK inhibitor U0126, 10 μM of the p38 MAPK inhibitor SB203580, or 10 μM of the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 for 1 hr, then treated with or without IFNγ + sCD40L for an additional 24 hr. Shown are the means of three separate experiments ± SEM. *P < 0.005, **P < 0.0008 compared with IFNγ + sCD40L. U0, U0126; LY, LY294002; SB, SB203580.

Fig. 3

IFNγ + sCD40L induces phosphorylation of ERK1/2, and treatment with U0126 abrogates phosphorylation. Western blot analysis of IFNγ + sCD40L-treated microglia shows phosphorylated ERK1/2 (upper panel) and total ERK1/2 expression (lower panel). Shown is a representative blot of two separate experiments. Lane 1: untreated; lane 2: U0126 10 μM; lane 3: IFNγ + sCD40L; lane 4: IFNγ + sCD40L + U0126.

Fig. 4

IFNγ + sCD40L-induced CXCL8 secretion is dependent on the transcription factors NFκB and AP-1, but not C/EBPβ. Microglia were serum free for 24 hr and then were treated for 4 hr, after which nuclear lysate was prepared. Gel shift analysis with specific probe sequence from NFκB or AP-1 was performed. Both the NFκB and the AP-1 probes demonstrated an increase in DNA-protein binding compared with the untreated. Shown is a representative blot of three separate experiments. Lanes 1 and 7: untreated (untx); lanes 2 and 8: IFNγ + sCD40L; lane 3: IFNγ + sCD40L + anti-p65; lane 4: IFNγ + sCD40L + anti-p50; lane 5: IFNγ + sCD40L + anti-p52; lane 6: IFNγ + sCD40L + cold NFκB oligonucleotide; lane 9: IFNγ + sCD40L + anti-ATF2; lane 10: IFNγ + sCD40L + anti-cJun; lane 11: cold AP-1 oligonucleotide.

Fig. 5

CXCL8 colocalizes with CD68/CD40-positive cells in HIV encephalitic brain tissue sections. Immunohistochemical analyses of human brain tissue sections with antibodies to CD68 (red staining), CD40 (green staining), and CXCL8 (cyan staining) were performed and analyzed by confocal microscopy. A–D: In normal brain tissue, there is minimal reactivity with all three antibodies. E–H: HIV tissue with no CNS compromise showed increased reactivity with antibodies, with some colocalization (merge, white staining). HIVE tissue with diffuse activated microglia (I–L) and a microglia nodule (M–P) demonstrated greater staining with antibodies and almost perfect colocalization of CXCL8 with CD68/CD40-positive cells (merge, white staining). Shown are representative figures from three separate tissue sections. Scale bar = 50 μm.