Intrathecal HIV-1 envelope glycoprotein gp120 induces enhanced pain states mediated by spinal cord proinflammatory cytokines

Abstract

Perispinal (intrathecal) injection of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein gp120 creates exaggerated pain states. Decreases in response thresholds to both heat stimuli (thermal hyperalgesia) and light tactile stimuli (mechanical allodynia) are rapidly induced after gp120 administration. gp120 is the portion of HIV-1 that binds to and activates microglia and astrocytes. These glial cells have been proposed to be key mediators of gp120-induced hyperalgesia and allodynia because these pain changes are blocked by drugs thought to affect glial function preferentially. The aim of the present series of studies was to determine whether gp120-induced pain changes involve proinflammatory cytokines [interleukin-1beta (IL-1) and tumor necrosis factor-alpha (TNF-alpha)], substances released from activated glia. IL-1 and TNF antagonists each prevented gp120-induced pain changes. Intrathecal gp120 produced time-dependent, site-specific increases in TNF and IL-1 protein release into lumbosacral CSF; parallel cytokine increases in lumbar dorsal spinal cord were also observed. Intrathecal administration of fluorocitrate (a glial metabolic inhibitor), TNF antagonist, and IL-1 antagonist each blocked gp120-induced increases in spinal IL-1 protein. These results support the concept that activated glia in dorsal spinal cord can create exaggerated pain states via the release of proinflammatory cytokines.

Fig. 1.

Blockade of intrathecal gp120-induced mechanical allodynia by intrathecal IL-1ra. Rats were assessed for low-threshold mechanical sensitivity (von Frey test) both before (BL) and 20–120 min after completion of intrathecal drug administration. Replicating our previous study (Milligan et al., 2000), intrathecal gp120 produced low-threshold mechanical allodynia in rats pretreated with the vehicle of IL-1ra (Vehicle + gp120;black squares), compared with controls (Vehicle + Vehicle; white squares). Although IL-1ra had no effect in the absence of gp120 (IL1ra + Vehicle;white circles), IL-1ra blocked mechanical allodynia induced by gp120 (IL1ra + gp120; black circles). i.t., Intrathecal.

Fig. 2.

Blockade of intrathecal gp120-induced thermal hyperalgesia by intrathecal IL-1ra. Rats were assessed for heat sensitivity (Hargreaves test) both before (BL) and 20–120 min after completion of intrathecal drug administration. Replicating our previous study (Milligan et al., 2000), intrathecal gp120 produced thermal hyperalgesia in rats pretreated with the vehicle of IL-1ra (Vehicle + gp120; black squares), compared with controls (Vehicle + Vehicle; white squares). Although IL-1ra had no effect in the absence of gp120 (IL1ra + Vehicle; white circles), IL-1ra blocked thermal hyperalgesia induced by gp120 (IL1ra + gp120; black circles).

Fig. 3.

Elevations of lumbar dorsal spinal cord IL-1 produced by intrathecal gp120 are blocked by pretreatment with IL-1ra. After completion of behavioral testing (see Figs. 1, 2), lumbar dorsal spinal cord was collected and assayed by ELISA for IL-1 protein. Relative to controls (Vehicle + Vehicle; white bar), intrathecal gp120 increased lumbar dorsal spinal cord IL-1 protein (Vehicle + gp120; black bar). Although IL-1ra had no effect in the absence of gp120 (IL1ra + Vehicle; herringbone bar), IL-1ra blocked the gp120-induced increase of IL-1 at this time (IL1ra + gp120; striped bar).

Fig. 4.

Time course of IL-1 protein changes in the dorsal spinal cord after lumbosacral intrathecal gp120 administration. Rats were administered intrathecal gp120 (black squares) or intrathecal vehicle (black-and-white squares) either 20, 40, 60, 90, or 120 min before tissue collection. A, gp120 increased IL-1 protein in the dorsal lumbar spinal cord, relative to vehicle controls. B, Site specificity of this effect was found based on the fact that increases in IL-1 protein in the cervical spinal cord were both much lower in magnitude and slower to occur. Note that the assay units in this figure are in picograms (see Fig. 5, units in nanograms).

Fig. 5.

Time course of IL-1 protein changes in lumbosacral CSF after lumbosacral intrathecal gp120 administration. Rats were administered intrathecal gp120 (black squares) or intrathecal vehicle (black-and-white square) either 20, 40, 60, 90, or 120 min before tissue collection. A, gp120 increased IL-1 protein in lumbosacral CSF, relative to vehicle controls. B, Site specificity of this effect was found based on the fact that increases in IL-1 protein in the cervical CSF were both much lower in magnitude and slower to occur. Note that the assay units in this figure are in nanograms versus picograms in Figure4.

Fig. 6.

Time course of TNF protein changes in lumbosacral CSF after lumbosacral intrathecal gp120 administration. Rats were administered intrathecal gp120 (black squares) or intrathecal vehicle (black-and-white squares) either 20, 40, 60, 90, or 120 min before tissue collection. A, gp120 increased TNF protein in lumbosacral CSF, relative to vehicle controls. B, Site specificity of this effect was found based on the fact that increases in TNF protein in cervical CSF were both much lower in magnitude and slower to occur.

Fig. 7.

Blockade of intrathecal gp120-induced mechanical allodynia by intrathecal TNFbp. Rats were assessed for low-threshold mechanical sensitivity (von Frey test) both before (BL) and 20–120 min after completion of intrathecal drug administration. Rats were administered either intrathecal TNFbp or vehicle before either intrathecal gp120 or vehicle. Replicating our previous work (Milligan et al., 2000) and Experiment 1, intrathecal gp120 produced mechanical allodynia in the absence of TNFbp (Vehicle + gp120;black squares), compared with controls (Vehicle + Vehicle; white squares). Although TNFbp had no effect in the absence of gp120 (TNFbp + vehicle;white circles), TNFbp partially blocked gp120-induced mechanical allodynia (TNFbp + gp120; black circles). ELISA results from these same animals are below (see Fig. 8).

Fig. 8.

Elevations of dorsal lumbar spinal cord IL-1 and lumbosacral CSF IL-1 produced by intrathecal gp120 are both blocked by pretreatment with intrathecal TNFbp. After completion of behavioral testing (see Fig. 7), dorsal lumbar spinal cord (A) and lumbosacral CSF (B) were collected and assayed by ELISA for IL-1 protein. Relative to controls (Vehicle + Vehicle; white bar), intrathecal gp120 increased dorsal lumbar spinal cord IL-1 protein content (Vehicle + gp120; black bar). Although TNFbp had no effect in the absence of gp120 (TNF-bp + Vehicle; herringbone bar), TNFbp blocked the gp120-induced increase of IL-1 at this time (TNF-bp + gp120; striped bar).

Fig. 9.

Elevations of dorsal lumbar spinal cord IL-1 and lumbosacral CSF IL-1 produced by intrathecal gp120 are both attenuated by pretreatment with intrathecal fluorocitrate. The behavioral data from these animals, which demonstrate that intrathecal fluorocitrate blocks gp120-induced pain states, have been published previously (Milligan et al., 2000). After completion of behavioral testing (Milligan et al., 2000), dorsal lumbar spinal cord (A) and lumbosacral CSF (B) were collected and assayed by ELISA for IL-1 protein. Relative to controls (Vehicle + Vehicle; white bar), intrathecal gp120 increased dorsal lumbar spinal cord IL-1 protein content (Vehicle + gp120; black bar). Although fluorocitrate had no effect in the absence of gp120 (Fluorocitrate + Vehicle; herringbone bar), fluorocitrate attenuated the gp120-induced increase of IL-1 at this time (Fluorocitrate + gp120; striped bar).

Fig. 10.

Astrocyte and microglial activation after gp120.A, C, Each graph was generated by analyzing every captured image at 0–100% threshold. By doing so, each image varied through the range of 100–0% of the field that was black. This allows the midrange of the functions to be determined for statistical analysis. The downward arrows in A andC indicate the point on the function that was statistically analyzed and graphically presented in Band D, respectively. B, Compared with vehicle controls (white bar), an increase in astrocyte activation (GFAP immunoreactivity) progressively occurred between 4 hr (black bar), 8 hr (herringbone bar), and 18 hr (striped bar) after intrathecal gp120.D, Increased microglial activation (OX-42 labeling) occurred after gp120 (time points as described above) compared with vehicle controls (white bar).

Fig. 11.

Photomicrographs of activation of dorsal lumbar spinal cord astrocytes by intrathecal gp120. A, C, GFAP labeling of lumbar dorsal horn at 8 and 18 hr, respectively, after intrathecal vehicle. B, D, GFAP labeling of lumbar dorsal horn at 8 and 18 hr, respectively, after intrathecal gp120. Scale bar, 50 μm. veh, Vehicle.

Fig. 12.

Photomicrographs of activation of dorsal lumbar spinal cord microglia by intrathecal gp120. A, C, OX-42 labeling of lumbar dorsal horn at 8 and 18 hr, respectively, after intrathecal vehicle. B, D, OX-42 labeling of lumbar dorsal horn at 8 and 18 hr, respectively, after intrathecal gp120. Scale bar, 50 μm.