Antinociception occurs with a reversal in alpha 2-adrenoceptor regulation of TNF production by peripheral monocytes/macrophages from pro- to anti-inflammatory

Abstract

Tumor necrosis factor-alpha (TNF) plays a role in neuropathic pain. During neuropathic pain development in the chronic constriction injury model, elevated TNF levels in the brain occur in association with enhanced alpha 2-adrenoceptor inhibition of norepinephrine release. alpha 2-Adrenoceptors are also located on peripheral macrophage where they normally function as pro-inflammatory, since they increase the production of the cytokine TNF, a proximal mediator of inflammation. How the central increase in TNF affects peripheral alpha 2-adrenoceptor function was investigated. Male, Sprague-Dawley rats had four loose ligatures placed around the right sciatic nerve. Thermal hyperalgesia was determined by comparing hind paw withdrawal latencies between chronic constriction injury and sham-operated rats. Chronic constriction injury increased TNF immunoreactivity at the lesion and the hippocampus. Amitriptyline, an antidepressant that is used as an analgesic, was intraperitoneally administered (10 mg/kg) starting simultaneous with ligature placement (day-0) or at days-4 or -6 post-surgery. Amitriptyline treatment initiated at day-0 or day-4 post-ligature placement alleviated hyperalgesia. When initiated at day-0, amitriptyline prevented increased TNF immunoreactivity in the hippocampus and at the lesion. A peripheral inflammatory response, macrophage production of TNF, was also assessed in the current study. Lipopolysaccharide (LPS)-stimulated production of TNF by whole blood cells and peritoneal macrophages was determined following activation of the alpha 2-adrenoceptor in vitro. alpha 2-Adrenoceptor regulation of TNF production from peripheral immune-effector cells reversed from potentiation in controls to inhibition in chronic constriction injured rats. This effect is accelerated with amitriptyline treatment initiated at day-0 or day-4 post-ligature placement. Amitriptyline treatment initiated day-6 post-ligature placement did not alleviate hyperalgesia and prevented the switch from potentiation to inhibition in alpha 2-adrenoceptor regulation of TNF production. Recombinant rat TNF i.c.v. microinfusion reproduces the response of peripheral macrophages from rats with chronic constriction injury. A reversal in peripheral alpha 2-adrenoceptor regulation of TNF production from pro- to anti-inflammatory is associated with effective alleviation of thermal hyperalgesia. Thus, alpha 2-adrenoceptor regulation of peripheral TNF production may serve as a potential biomarker to evaluate therapeutic regimens.

Fig. 1

A proposed interactive relationship between levels of TNF and α₂-adrenergic autoreceptor (α₂-AR) coupling to specific G-proteins directs neurotransmitter release. Changes in the production of TNF affect G-protein expression that, in turn, directs G-protein coupling of presynaptic receptors that direct norepinephrine release. For example, an increase in TNF increases Gα_i-proteins, while decreasing TNF has the opposite effect. Thus, an increase in Gα_i-proteins favors α₂-AR-Gα_i-protein coupling when TNF levels are high, while a decrease in Gα_i-proteins favors α₂-AR-Gα_s-protein coupling when TNF levels are low. The α₂-AR normally favors coupling to the Gα_i subunit, and activation of these inhibitory autoreceptors decreases norepinephrine release from noradrenergic neurons. Activation of the α₂-AR coupled to Gα_i also inhibits TNF production in the brain. Because TNF normally inhibits norepinephrine release, this reduction in levels of TNF increases norepinephrine release, thus maintaining homeostasis of the levels of bioactive norepinephrine. However, decreases in the levels of TNF switch the functioning of the α₂-AR. The α₂-AR now exists predominantly as a stimulatory autoreceptor (designated as α₂-AR coupled to Gα_s), and its activation both facilitates norepinephrine release and increases TNF production. This system remains in check since increased levels of TNF support coupling of the α₂-AR to the Gα_i subunit. These events continually occur within an equilibrium by which physiologic levels of TNF and normal functioning of the α₂-AR are preserved. A pathologic increase in TNF production during chronic pain disrupts the system’s natural balance, such that increased TNF levels are sustained and norepinephrine release remains low, while the α₂-AR undergoes a dysfunctional adaptation, disproportionately favoring one functional form (couples to Gα_i) over the other (coupling to Gα_s) (favoring the ‘left’ or shaded side of model). Conversely, decreasing TNF levels perturbs the system in the opposite fashion; increasing norepinephrine release and returning the α₂-AR to a state of functional balance. This is a proposed mechanism by which TNF levels and the α₂-AR participate in a reciprocal fashion in the pathogenesis of chronic pain (also chronic stress or depression) and in the therapeutic efficacy of antidepressant drugs. While a switch in α₂-AR-G-protein coupling supports the interactive relationship between levels of TNF and α₂-AR regulation of norepinephrine release, a switch in α-AR phenotype (α₂ to α₁) may also occur. α₂-AR = α₂-adrenoceptor; NE = norepinephrine, TNF = tumor necrosis factor-α.

Fig. 2

Assessment of thermal hyperalgesia in rats undergoing chronic constriction injury alone, or with concomitant treatment with amitriptyline. Rats receive ligature placement unilaterally around the sciatic nerve, alone, or with concomitant treatment with amitriptyline (10 mg/kg, i.p., twice daily). Data are presented as the difference score of ipsilateral (experimental) – contralateral (control) hind paw withdrawal latency in seconds. Each point is expressed as the mean ± S.E.M. (number of rats in brackets). (A) Amitriptyline treatment is initiated 1 h prior to ligature placement (indicated by the arrow), and continued every 12 h until pre-determined times post-ligature placement. Note: Amitriptyline treatment concomitant with ligature placement delays and attenuates thermal hyperalgesia. Statistical significance analyzed by two-way ANOVA followed by Tukey multiple comparison tests ^* P < 0.05, ^** P < 0.001 compared to sham-operated; # P < 0.001, compared to chronic constriction injury rats. (B) Amitriptyline treatment is initiated at day-4 post-ligature placement, indicated by the position of the arrow, in rats receiving unilateral ligature placement around the sciatic nerve. Statistically significant as analyzed using one-way ANOVA followed by Dunnett’s analysis with CCI-SAL(0) rats serving as the control for comparisons, ^* P < 0.05. Note: Amitriptyline treatment initiated on day-4 post-ligature placement rapidly attenuates thermal hyperalgesia. (C) Amitriptyline treatment is initiated in rats receiving unilateral ligature placement around the sciatic nerve at day-6 post-ligature placement, indicated by the position of the arrow. Statistically significant as analyzed using one-way ANOVA followed by Dunnett’s analysis with CCI-SAL(0) rats serving as the control for comparisons, ^* P < 0.05. Note: When initiated at day-6 post-ligature placement, amitriptyline is no longer efficacious in treating thermal hyperalgesia in the chronic constriction injury model of neuropathic pain (compare to panel 2A). SHAM-SAL(0) = sham surgery of the sciatic nerve with simultaneous saline administration; SHAM-AMT(0) = sham surgery of the sciatic nerve with simultaneous amitriptyline administration; CCI-AMT(0) = amitriptyline treatment concomitant with chronic constriction injury; CCI-SAL(0) = chronic constriction injury with simultaneous saline administration; CCI-AMT(4) = amitriptyline treatment initiated at day-4 post-ligature placement; CCI-AMT(6) = amitriptyline treatment initiated at day-6 post-ligature placement.

Fig. 2

Assessment of thermal hyperalgesia in rats undergoing chronic constriction injury alone, or with concomitant treatment with amitriptyline. Rats receive ligature placement unilaterally around the sciatic nerve, alone, or with concomitant treatment with amitriptyline (10 mg/kg, i.p., twice daily). Data are presented as the difference score of ipsilateral (experimental) – contralateral (control) hind paw withdrawal latency in seconds. Each point is expressed as the mean ± S.E.M. (number of rats in brackets). (A) Amitriptyline treatment is initiated 1 h prior to ligature placement (indicated by the arrow), and continued every 12 h until pre-determined times post-ligature placement. Note: Amitriptyline treatment concomitant with ligature placement delays and attenuates thermal hyperalgesia. Statistical significance analyzed by two-way ANOVA followed by Tukey multiple comparison tests ^* P < 0.05, ^** P < 0.001 compared to sham-operated; # P < 0.001, compared to chronic constriction injury rats. (B) Amitriptyline treatment is initiated at day-4 post-ligature placement, indicated by the position of the arrow, in rats receiving unilateral ligature placement around the sciatic nerve. Statistically significant as analyzed using one-way ANOVA followed by Dunnett’s analysis with CCI-SAL(0) rats serving as the control for comparisons, ^* P < 0.05. Note: Amitriptyline treatment initiated on day-4 post-ligature placement rapidly attenuates thermal hyperalgesia. (C) Amitriptyline treatment is initiated in rats receiving unilateral ligature placement around the sciatic nerve at day-6 post-ligature placement, indicated by the position of the arrow. Statistically significant as analyzed using one-way ANOVA followed by Dunnett’s analysis with CCI-SAL(0) rats serving as the control for comparisons, ^* P < 0.05. Note: When initiated at day-6 post-ligature placement, amitriptyline is no longer efficacious in treating thermal hyperalgesia in the chronic constriction injury model of neuropathic pain (compare to panel 2A). SHAM-SAL(0) = sham surgery of the sciatic nerve with simultaneous saline administration; SHAM-AMT(0) = sham surgery of the sciatic nerve with simultaneous amitriptyline administration; CCI-AMT(0) = amitriptyline treatment concomitant with chronic constriction injury; CCI-SAL(0) = chronic constriction injury with simultaneous saline administration; CCI-AMT(4) = amitriptyline treatment initiated at day-4 post-ligature placement; CCI-AMT(6) = amitriptyline treatment initiated at day-6 post-ligature placement.

Fig. 2

Assessment of thermal hyperalgesia in rats undergoing chronic constriction injury alone, or with concomitant treatment with amitriptyline. Rats receive ligature placement unilaterally around the sciatic nerve, alone, or with concomitant treatment with amitriptyline (10 mg/kg, i.p., twice daily). Data are presented as the difference score of ipsilateral (experimental) – contralateral (control) hind paw withdrawal latency in seconds. Each point is expressed as the mean ± S.E.M. (number of rats in brackets). (A) Amitriptyline treatment is initiated 1 h prior to ligature placement (indicated by the arrow), and continued every 12 h until pre-determined times post-ligature placement. Note: Amitriptyline treatment concomitant with ligature placement delays and attenuates thermal hyperalgesia. Statistical significance analyzed by two-way ANOVA followed by Tukey multiple comparison tests ^* P < 0.05, ^** P < 0.001 compared to sham-operated; # P < 0.001, compared to chronic constriction injury rats. (B) Amitriptyline treatment is initiated at day-4 post-ligature placement, indicated by the position of the arrow, in rats receiving unilateral ligature placement around the sciatic nerve. Statistically significant as analyzed using one-way ANOVA followed by Dunnett’s analysis with CCI-SAL(0) rats serving as the control for comparisons, ^* P < 0.05. Note: Amitriptyline treatment initiated on day-4 post-ligature placement rapidly attenuates thermal hyperalgesia. (C) Amitriptyline treatment is initiated in rats receiving unilateral ligature placement around the sciatic nerve at day-6 post-ligature placement, indicated by the position of the arrow. Statistically significant as analyzed using one-way ANOVA followed by Dunnett’s analysis with CCI-SAL(0) rats serving as the control for comparisons, ^* P < 0.05. Note: When initiated at day-6 post-ligature placement, amitriptyline is no longer efficacious in treating thermal hyperalgesia in the chronic constriction injury model of neuropathic pain (compare to panel 2A). SHAM-SAL(0) = sham surgery of the sciatic nerve with simultaneous saline administration; SHAM-AMT(0) = sham surgery of the sciatic nerve with simultaneous amitriptyline administration; CCI-AMT(0) = amitriptyline treatment concomitant with chronic constriction injury; CCI-SAL(0) = chronic constriction injury with simultaneous saline administration; CCI-AMT(4) = amitriptyline treatment initiated at day-4 post-ligature placement; CCI-AMT(6) = amitriptyline treatment initiated at day-6 post-ligature placement.

Fig. 3

Caliper measurements of sciatic nerve diameter in rats undergoing chronic constriction injury, alone or with concomitant amitriptyline treatment: Data are presented as the difference of ipsilateral (experimental) – contralateral (control) nerve diameter (mm). Each point is expressed as the mean ± S.E.M. (number of rats in brackets). Inset shows the difference values in appropriate control rats. CCI-SAL(0) = chronic constriction injury with simultaneous saline administration; CCI-AMT(0) = amitriptyline treatment concomitant with ligature placement; CCI-AMT(4) = amitriptyline treatment initiated at day-4 post-ligature placement; CCI-AMT(6) = amitriptyline treatment initiated at day-6 post-ligature placement. Note: Compared to controls (inset), chronic constriction injury causes increases in the diameter of the ipsilateral sciatic nerves at all times post-ligature placement examined (P < 0.05, ANOVA on Ranks followed by Dunn’s post-hoc analysis). Amitriptyline treatment, regardless of time of treatment initiation, has no effect on chronic constriction injury -induced increases in ipsilateral nerve diameters, and therefore, on the difference values.

Fig. 4

Immunoreactive staining for TNF in ipsilateral sciatic nerves from day-8 rats. (A) Shown in each panel is a representative longitudinal section of sciatic nerve of three independent animals: (a) Sham-8 rats; (b) rats at day-8 post-ligature placement (CCI-8); (c) rats at day-8 post-ligature placement with amitriptyline (10 mg/kg, i.p., twice daily) treatment initiated at day-0 post-ligature placement (CCI-8-AMT(0)); (d) rats at day-8 post-ligature placement with amitriptyline treatment initiated at day-4 post-ligature placement (CCI-8-AMT(4)); and (e) rats at day-8 post-ligature placement with amitriptyline treatment initiated at day-6 post-ligature placement (CCI-8-AMT(6)). Scale bar = 50 µM (B) Quantitative analysis of TNF immunoreactive staining. Statistical significance was determined using one-way ANOVA followed by Dunnett’s analysis of mean density values with CCI-8 serving as the control for all comparisons: Sham-8 vs. CCI-8 (P < 0.05); CCI-8-AMT(0) vs. CCI-8 (P < 0.05). For each group, n=3.

Fig. 4

Immunoreactive staining for TNF in ipsilateral sciatic nerves from day-8 rats. (A) Shown in each panel is a representative longitudinal section of sciatic nerve of three independent animals: (a) Sham-8 rats; (b) rats at day-8 post-ligature placement (CCI-8); (c) rats at day-8 post-ligature placement with amitriptyline (10 mg/kg, i.p., twice daily) treatment initiated at day-0 post-ligature placement (CCI-8-AMT(0)); (d) rats at day-8 post-ligature placement with amitriptyline treatment initiated at day-4 post-ligature placement (CCI-8-AMT(4)); and (e) rats at day-8 post-ligature placement with amitriptyline treatment initiated at day-6 post-ligature placement (CCI-8-AMT(6)). Scale bar = 50 µM (B) Quantitative analysis of TNF immunoreactive staining. Statistical significance was determined using one-way ANOVA followed by Dunnett’s analysis of mean density values with CCI-8 serving as the control for all comparisons: Sham-8 vs. CCI-8 (P < 0.05); CCI-8-AMT(0) vs. CCI-8 (P < 0.05). For each group, n=3.

Fig. 5

Immunoreactivity for TNF in contralateral hippocampal sections. (A) Shown in each panel is a representative coronal section of hippocampus of four independent animals from: (a) Sham-operated (day-8 post-surgery), (b) Day-8 post-ligature placement (CCI-8), and (c) Day-8 post-ligature placement with concomitant (initiated at day-0) amitriptyline treatment (10 mg/kg, i.p., twice daily) (CCI-8-AMT(0)). Scale bar = 50 µM (B) Quantitative analysis of TNF immunoreactive staining. Statistical analysis of mean density values (one-way ANOVA followed by Tukey test) demonstrates that TNF immunoreactivity is significantly enhanced in the contralateral hippocampus at day- 8 post-ligature placement as compared to the contralateral hippocampus from sham-operated rats (P < 0.01). Concomitant treatment with amitriptyline prevents the increase in TNF immunoreactivity in the hippocampus (NS, as compared to sham-operated). For each group, n=4.

Fig. 5

Immunoreactivity for TNF in contralateral hippocampal sections. (A) Shown in each panel is a representative coronal section of hippocampus of four independent animals from: (a) Sham-operated (day-8 post-surgery), (b) Day-8 post-ligature placement (CCI-8), and (c) Day-8 post-ligature placement with concomitant (initiated at day-0) amitriptyline treatment (10 mg/kg, i.p., twice daily) (CCI-8-AMT(0)). Scale bar = 50 µM (B) Quantitative analysis of TNF immunoreactive staining. Statistical analysis of mean density values (one-way ANOVA followed by Tukey test) demonstrates that TNF immunoreactivity is significantly enhanced in the contralateral hippocampus at day- 8 post-ligature placement as compared to the contralateral hippocampus from sham-operated rats (P < 0.01). Concomitant treatment with amitriptyline prevents the increase in TNF immunoreactivity in the hippocampus (NS, as compared to sham-operated). For each group, n=4.

Fig. 6

α₂-Adrenoceptor regulation of TNF production from peritoneal macrophages harvested from rats undergoing paradigms indicated under the abscissa. Data are expressed as % change in LPS (30 ng/ml)-stimulated TNF production with clonidine (10⁻⁷ M) in vitro calculated with respect to macrophages stimulated with LPS alone. Culture supernatants were harvested after 4 h, and analyzed for biologically active TNF as explained in Materials and Methods. The n values for each group are indicated in brackets on top of the respective bars. (A) α₂-Adrenoceptor regulation of TNF production from macrophages harvested from rats undergoing chronic constriction injury, in comparison to control group. The control group (n = 8) consists of data pooled from naïve (4) and Sham-8 (4) animals, since no significant differences were observed among the two groups (TNF (pg/ml) in supernatants from non-stimulated macrophages: naïve, 14.7 ± 5.5 pg/ml versus Sham-8, 15.6 ± 1.4 pg/ml, NS; or stimulated with 30 ng/ml LPS alone: naïve, 49.4 ± 23.8 pg/ml versus Sham-8, 116.5 ± 78.3 pg/ml, NS, Student’s t-test). Statistically significant ^* P < 0.05, ^** P < 0.001, as compared to the control group, Student’s t-test. (B) Effect of simultaneous treatment of chronic constriction injury-rats with amitriptyline (10 mg/kg, i.p., twice daily) on subsequent in vitro α₂-adrenergic regulation of LPS (30 ng/ml)-stimulated TNF production. Statistically significant ^# P = 0.01, as compared to day-4 post-ligature placement group, Student’s t-test. (C) Comparison of amitriptyline administration (10 mg/kg, i.p., twice daily) alone or simultaneous with chronic constriction injury on α₂-adrenoceptor regulation of macrophage-derived TNF in vitro. Statistically significant ^# P < 0.01, as compared to control, one-way ANOVA run at each time frame followed by Tukey test. (D) α₂-Adrenoceptor regulation of LPS-stimulated, macrophage-derived TNF production from rats terminated at day-8 post-ligature placement. Statistically significant ^† P < 0.02, as compared to day-8 post-ligature placement (CCI-8) group, Student’s t-test. (E) α₂-Adrenoceptor regulation of LPS-stimulated, macrophage-derived TNF production from rats terminated at day-16 post-ligature placement. Statistically significant ^† P < 0.02, ^* P < 0.05, as compared to day-16 post-ligature placement (CCI-16) group, Student’s t-test. CONT = control; AMT = amitriptyline; CCI = chronic constriction injury; CCI-AMT(0) = amitriptyline treatment initiated concomitant with ligature placement.

Fig. 7

(A) Effect of in vitro α₂-adrenoceptor activation (clonidine, 10⁻⁷ M) on LPS (30 ng/ml)-stimulated TNF production in whole blood cultures harvested from rats undergoing chronic constriction injury alone or with simultaneous amitriptyline (10 mg/kg, i.p., twice daily) treatment. Statistically significant ^* P < 0.05 as compared to ‘control’, one-way ANOVA followed by Dunnett’s post-hoc test run at each time frame post-ligature placement. The control group (n = 6) consists of data pooled from naïve (4) and Sham-8 (2) animals, based on no observable differences among the whole blood ‘control’ groups (TNF (pg/ml) in supernatants from non-stimulated whole blood cultures: naïve, 4.0 ± 0.9 pg/ml versus Sham-8, 9.7 ± 7.6 pg/ml; or whole blood cultures stimulated with 30 ng/ml LPS alone: naïve, 15.6 ± 4.3 pg/ml versus Sham-8, 23.5 ± 6.3 pg/ml). (B) Effect of α₂-adrenoceptor activation on LPS-stimulated TNF production in vitro in whole blood cultures harvested from rats at day-8 post-ligature placement. Statistically significant ^* P < 0.05 as compared to amitriptyline administration initiated at day-0, one-way ANOVA followed by Dunnett’s post-hoc test with CCI-8 alone serving as the control for all comparisons. CCI = chronic constriction injury; CCI-AMT(0) = CCI-rats receiving concomitant treatment with amitriptyline (10 mg/kg, i.p., twice daily). The number of determinations for each paradigm is indicated in brackets above the bars.

Fig. 8

Effect of in vitro α₂-adrenoceptor activation (clonidine, 10⁻⁷ M) on LPS (30 ng/ml)-stimulated TNF production in peritoneal macrophages harvested from rats infused with rrTNF into the right lateral cerebral ventricle for 14 days. Data are expressed as the % change in TNF levels released into supernatants as compared to LPS stimulation alone. Statistical significance of % change in TNF levels compared to aCSF-infused rats ^* P < 0.05 was determined using Student’s t-test. Each bar represents the mean ± S.E.M. with the number of rats indicated in brackets.