Chemokines and glycoprotein120 produce pain hypersensitivity by directly exciting primary nociceptive neurons

Abstract

Human immunodeficiency virus-1 (HIV-1) infection is associated with numerous effects on the nervous system, including pain and peripheral neuropathies. We now demonstrate that cultured rat dorsal root ganglion (DRG) neurons express a wide variety of chemokine receptors, including those that are thought to act as receptors for the HIV-1 coat protein glycoprotein120 (gp120). Chemokines that activate all of the known chemokine receptors increased [Ca(2+)](i) in subsets of cultured DRG cells. Many neurons responded to multiple chemokines and also to bradykinin, ATP, and capsaicin. Immunohistochemical studies demonstrated the expression of the CXCR4 and CCR4 chemokine receptors on populations of DRG neurons that also expressed substance P and the VR1 vanilloid receptor. RT-PCR analysis confirmed the expression of CXCR4, CX3CR1, CCR4, and CCR5 mRNAs in DRG neurons. Chemokines and gp120 produced excitatory effects on DRG neurons and also stimulated the release of substance P. Chemokines and gp120 also produced allodynia after injection into the rat paw. Thus these results provide evidence that chemokines and gp120 may produce painful effects via direct actions on chemokine receptors expressed by nociceptive neurons. Chemokine receptor antagonists may be important therapeutic interventions in the pain that is associated with HIV-1 infection and inflammation.

Fig. 1.

Chemokines and gp120s increased [Ca²⁺]_i in single cultured rat DRG neurons. This figure illustrates examples of responses to individual applications of several chemokines. The complete list of chemokines tested in this paradigm is provided in Table 1. gp120 SIV_mac251 selective for CCR5, gp120 HIV-1_IIIBselective for CXCR4, and chemokines that activate different CCRs (RANTES, MDC, TARC, eotaxin, I-309), CXCR4 (SDF-1α) as well as CX3CR1 (fractalkine) were applied for the time indicated by thebars (3 min). Concentrations of chemokines that were applied included RANTES (50 nm), gp120 SIV_mac251 (200 pm), SDF-1α (50 nm), fractalkine (100 nm), gp120 HIV-1_IIIB (200 pm), MDC (50 nm), I-309 (50 nm), eotaxin (50 nm), and TARC (50 nm).

Fig. 2.

DRG neurons exhibited complex patterns of responsiveness to chemokines in a fura-2-based calcium-imaging experiment. This figure shows examples of responsiveness of individual DRG neurons to combinations of chemokines. Note that chemokine-responsive neurons were also frequently responsive to known excitants of nociceptive neurons such as bradykinin, capsaicin, or ATP. However, this was not inevitably the case (e.g., compareB, C). Concentrations of molecules that were applied in these studies were eotaxin (E; 50 nm), MDC (50 nm), TARC (50 nm), MCP-1 (50 nm), MIP-1β (50 nm), IP-10 (50 nm), SLC (50 nm), BCA (50 nm), VMIP-I (v-I, 100 nm), VMIP-II (v-II, 100 nm), VMIP-III (v-III, 100 nm), bradykinin (BK, 1 μm), capsaicin (Cap, 1 μm), and ATP (100 μm).

Fig. 3.

Chemokines and gp120s excite DRG neurons. A, Illustrated are the effects of the chemokine MDC on the neuronal spike threshold. The neuron was unable to fire an action potential when 5 pA of current was injected under normal conditions (a) but did so, in a reversible manner, 2 min after the addition of MDC (b). Note that MDC did not change the membrane potential. Note also that this neuron was excited by capsaicin (d), indicating its identity as a nociceptive neuron. c, e, Illustrated is the washout of the effects of MDC and capsaicin, respectively.B, In this case the addition of gp120 lowered the spike threshold in this neuron, again without depolarizing the neuron (a, control; b, 2 min after the addition of gp120 SIV_mac251; c, after washout of the gp120). C, In some neurons, such as this example, gp120s or chemokines produced clear depolarization of the cell. Note that this neuron also was depolarized by capsaicin and bradykinin. A total of 20 neurons of 79 that were tested exhibited excitatory responses (MDC, 3 of 7; fractalkine, 1 of 7; SDF-1α, 1 of 7; eotaxin, 3 of 8; VMIP-2, 1 of 7; TARC, 1 of 7; gp120 SIV_mac251, 5 of 16; gp120 HIV-1_IIIB, 4 of 14; RANTES, 1 of 7). In these cells the chemokines or gp120 lowered the threshold without changing the membrane potential (45%; n = 9) or depolarized DRG neurons (55%; n = 11). Of the chemokine- or gp120-responsive neurons 70% (n = 14) also responded to capsaicin. Concentrations of chemokines that were applied included MDC (50 nm), fractalkine (100 nm), eotaxin (50 nm), VMIP-II (100 nm), TARC (50 nm), gp120 SIV_mac251 (200 pm), gp120 HIV-1_IIIB (200 pm), capsaicin (1 μm), and BK (1 μm).

Fig. 4.

Confocal laser microscopy analysis of CXCR4 expression on HEK 293 cells and DRG neurons. A, Illustrated is immunohistochemical staining for the cloned rat CXCR4 receptor expressed in HEK 293 cells (a). Also illustrated are controls in which the primary antibody was not applied (b) or HEK 293 cells in which the receptor was not expressed (c). B, Staining of cultured rat DRG cells revealed a population of neurons that expressed the CXCR4 receptor (a). Note the staining of both the cell soma and terminal varicosities. Staining for the CXCR4 receptor could be blocked by preabsorbing the antiserum with the peptide epitope against which it was raised (b).C, Colocalization of substance P and the CXCR4 receptor in rat DRG neurons. a, DRG neuron staining for substance P. b, Two DRG neurons staining for the CXCR4 receptor.c, Overlay of images in a andb showing the colocalization of substance P and the CXCR4 receptor in one neuron (black arrow) and the absence of substance P (white arrow) in the second CXCR4-stained neuron. D, Colocalization of VR1 and the CXCR4 receptor in rat DRG neurons. a, DRG neuron staining for VR1. b, Staining for CXCR4. c, Overlay of images in a and b showing the colocalization of VR1 and the CXCR4 receptor.

Fig. 5.

Confocal laser microscopy analysis of CCR4 expression on DRG neurons. A, Staining with anti-mouse CCR4 antiserum demonstrated the expression of CCR4 receptor in a population of neurons from cultured rat DRG cells. B, Colocalization of substance P and the CCR4 receptor in rat DRG neurons.a, DRG neuron staining for substance P.b, Staining for CCR4. c, Overlay of images in a and b showing the colocalization of substance P and the CCR4 receptor. C, Colocalization of VR1 and the CCR4 receptor in rat DRG neurons.a, DRG neuron staining for VR1. Three DRG neurons were stained positively for the VR1. b, Staining for CCR4.c, Overlay of images in a andb showing the colocalization of VR1 and the CCR4 receptor in one neuron (black arrow) and absence of CCR4 (white arrow) in the second VR1-stained neuron.D, Left, The effect of MDC, RANTES, gp120 HIV-1_IIIB, and bradykinin on substance P release from cultured neonatal DRG neurons. D,Right, The effects of 50 mmK⁺ and 1 μm capsaicin used as positive controls. *p < 0.05; **p < 0.01.

Fig. 6.

RT-PCR analysis of chemokine receptor expression on neonatal DRG neurons. Results demonstrate the presence of mRNA of CXCR4, CX3CR1, CCR5, and CCR4 in DRG neurons. Lanes 2and 3 in each panel show PCR products obtained from amplification by primers selected specifically to detect each chemokine receptor (lane 2, DRG neurons; lane 3, rat brain). Lane 1 contains 1 kb plus ladder (Life Technologies); lane 4 indicates no amplification products with H₂O.

Fig. 7.

Chemokines and coat proteins produce tactile allodynia. A, The intradermal administration of 500 ng of bradykinin (●), but not the vehicle control (○), reduced the threshold to withdraw the hindpaw in response to punctate mechanical stimuli. B, The intradermal administration of 250 ng of the chemokines RANTES (■), SDF-1α (▪), and MDC (○) or the coat proteins gp120 SIV_mac251 (▵) and gp120 HIV-1_IIIB (▴) also reduced the mechanical threshold.BL, The mean baseline threshold before the intradermal injection. The symbols represent the mean ± SEM from five to seven rats. Threshold values for all agents were significantly different from the corresponding time point in vehicle-treated rats; p < 0.01. These data were obtained concurrently and are presented in two panels for clarity of presentation.