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. 2008 Feb;15(3):183-90.
doi: 10.1038/sj.gt.3303054. Epub 2007 Nov 22.

HSV-mediated transfer of interleukin-10 reduces inflammatory pain through modulation of membrane tumor necrosis factor alpha in spinal cord microglia

Z Zhou  1 , X Peng, S Hao, D J Fink, M Mata
Affiliations

HSV-mediated transfer of interleukin-10 reduces inflammatory pain through modulation of membrane tumor necrosis factor alpha in spinal cord microglia

Z Zhou et al. Gene Ther. 2008 Feb.

Abstract

To dissect the molecular basis of the neuroimmune response associated with the genesis of inflammatory (nociceptive) pain, we constructed a herpes simplex virus-based gene transfer vector to express the antiinflammatory cytokine interleukin-10 (IL-10), and used it to examine the effect of IL-10 expression in activated microglial cells in vitro, and in inflammatory pain in vivo. IL-10 reduced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and decreased the expression of full-length membrane spanning tumor necrosis factor-alpha (mTNFalpha) following lipopolysaccharide stimulation of microglia in vitro. IL-10 also reduced intracellular cleavage of mTNFalpha and release of the soluble cleavage product sTNFalpha. Similar effects on TNFalpha expression were observed when the cells were pretreated with a p38 MAPK inhibitor. In animals, injection of a dilute solution of formalin in the skin resulted in an increase in mTNFalpha in spinal dorsal horn, without detectable sTNFalpha. Local release of IL-10 achieved by gene transfer reduced the number of spontaneous flinches in the early and delayed phases of the formalin test of inflammatory pain. The effect of IL-10 on nocisponsive behavior correlated with a block in phosphorylation of p38 and reduced expression of 26 kDa mTNFalpha in spinal microglia. The results emphasize the key role played by membrane TNFalpha in the spinal neuroimmune response in pain caused by peripheral inflammation.

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Conflict of interest statement

Disclosure/conflict of interest

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Schematic representation of the vector constructs QHIL10 (a) and QHGFP (b). Vector QHIL10 contains two copies of the rat interleukin-10 (IL-10) coding sequence in a nonreplicating HSV backbone, defective in expression of the HSV IE genes ICP4, ICP22, ICP27 and ICP47. Control vector QHGFP contains two copies of the green fluorescent protein (GFP) gene in the same HSV backbone.
Figure 2
Figure 2
(a) Primary DRG neurons 48 h after infection by QHGFP or QHIL10 (MOI of 1 for 2 h) stained for the neuronal marker Tuj1 (green) and interleukin-10 (IL-10, red). Scale bar = 10 μm. Western blot of cell lysate (b) and culture medium (c) 48 h after transfection at MOI of 1. (d) ELISA of IL-10 from the medium collected 48 h after transfection. Western blot of protein from DRG (e) and dorsal quadrant of spinal cord (f) 1 week after subcutaneous inoculation of QHIL10 or QHGFP. The identity of IL-10 as vector-derived is confirmed by the anti-HA antibody employed in the western blot.
Figure 3
Figure 3
(a) HAPI cells immunostained with the microglial cell marker OX42 (green). (b) HAPI cells immunostained with an antibody against the interleukin-10 (IL-10) receptor (red) with Hoechst (blue) counterstaining. (c) ELISA of culture medium collected 48 h after infection of HAPI cells with QHIL10 or QHGFP (MOI of 1 for 2 h). (d) Western blot of HAPI cell lysate 48 h after infection with QHIL10 or QHGFP. Scale bar = 10 μm.
Figure 4
Figure 4
Tumor necrosis factor-α (TNFα) mRNA and protein in HAPI cells transduced with QHGFP or QHIL10 and exposed to lipopolysaccharide (LPS) determined by reverse transcription (RT)–PCR (a) and western blot (c). The amount of mRNA was quantified by relative optical density (b) and the sum of 17 and 26 kDa protein bands quantified by chemiluminescence (d), normalized to β-actin mRNA and protein levels, respectively. The amount of sTNFα released into the culture medium was determined by ELISA (DY510, R&D Systems; e). The data represent the results of three independent experiments. Mean±s.e.m.; **P<0.01.
Figure 5
Figure 5
Tumor necrosis factor-α (TNFα) mRNA and TNFα protein (mTNFα plus sTNFα) in HAPI cells pretreated with 10 μM SB202190 for 30 min prior to addition of lipopolysaccharide (LPS) determined by RT–PCR (a) and western blot (c). The amount of mRNA was quantified by relative optical density (b) and the sum of the 17 and 26 kDa protein bands quantified by chemiluminescence (d), normalized to β-actin mRNA and protein levels, respectively. sTNFα released in the culture medium by ELISA (e). p-p38 and p38 protein (western blot) in HAPI cells transduced with QHGFP or QHIL10 and exposed the LPS (f). Ratio of p-p38 to p38-determined chemiluminescence (g). Data presented represent the results of three independent experiments. Mean±s.e.m.; *P<0.05; **P<0.01.
Figure 6
Figure 6
(a) Spontaneous flinching observed after injection of 50 μl of 5% formalin in animals 10 days after subcutaneous inoculation of QHIL10, QHGFP or phosphate-buffered saline (PBS). P<0.05 comparing QHIL10 to QHGFP or PBS by repeated measures analysis. Means±s.e.m., n = 8 animals per group. (b) Total number of flinches in phase 1 (1–10 min). (c) Total number of flinches in phase 2 (10–60 min). Mean±s.e.m., n = 8 animals per group, **P<0.01.
Figure 7
Figure 7
Tumor necrosis factor-α (TNFα) mRNA and protein in the dorsal quadrant of lumbar spinal cord determined by RT–PCR (a) and western blot (c), quantitated by relative optical density (b) and chemiluminescence (d), normalized to β-actin levels. Levels of p-p38 in the dorsal quadrant of lumbar spinal cord determined by western blot (e) and quantitated by relative optical density (f) normalized to total p38 levels. The data presented represent mean±s.e.m., n = 5 animals per group; *P<0.05, **P<0.01. (g) Double-label immunostaining of TNFα (red) and OX42 (green) in laminae I–II of dorsal horn (left, bar = 40 μm). Higher power magnification of individual microglial cells (left two panels, bar = 40 μm).
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