Recent investigations suggest that the sympathetic nervous system affects the immune system. This study examined whether stellate ganglion block (SGB) affects the immune response, specifically the distribution of lymphocyte subsets and natural-killer (NK) cell activity.
Ten volunteers received three different treatments in random order at 7-day intervals: (1) SGB with 7 ml of 1% lidocaine; (2) an identical volume of normal saline injected at the same site as SGB; and (3) an identical volume of 1% lidocaine injected intramuscularly. Blood samples were drawn before and 30 min after treatment. The distribution of lymphocyte subsets was analyzed, and NK cell activity was measured. Plasma concentrations of epinephrine, norepinephrine, adrenocorticotropic hormone, and cortisol were measured.
Any value in the normal saline and intramuscular treatments did not change significantly. After SGB, the plasma concentrations of epinephrine and norepinephrine decreased significantly (P < 0.01), but adrenocorticotropic hormone and cortisol values were unchanged. Increases were observed in the proportion of B cells (from 18.4 +/- 3.0% to 20.0 +/- 3.8%; P < 0.01) and T cells (from 64.2 +/- 4.1% to 67.1 +/- 4.2%; P < 0.01). A decrease in the proportion of NK cells was observed (from 13.4 +/- 2.7% to 9.8 +/- 2.2%; P < 0.01). The proportion of CD4+ cells increased (P < 0.01), and that of CD8+ cells decreased (P < 0.01), so that the CD4+ cell/CD8+ cell ratio increased (P < 0.01). The proportion of CD29+ (helper-inducer T) cells increased (P < 0.05), but that of CD45RA+ (suppressor-inducer T) cells did not change. NK cell activity decreased significantly (from 33.6 +/- 8.3% to 29.1 +/- 7.6%; P < 0.01).
A small but significant alteration in lymphocyte subsets and in NK cell activity by SGB indicates that local sympathetic nerve block may modulate the immune response.
THE hypothalamic-pituitary-adrenal and hypothalamic-sympathetic-adrenal axes are two important pathways that modify the immune response. Recently, not only hormonal but also neural–immune interactions have been reported, indicating that the sympathetic nervous system may play an important role in the immune response. For example, lymphoid organs are innervated extensively by noradrenergic sympathetic nerve fibers, and lymphocytes bear functional adrenoreceptors. 1–3Adrenergic agonists can modulate immune responses in vitro , including cytokine production, lymphocyte proliferation, and antibody secretion. 4–6Chemical sympathectomy can change the immune response in vivo . 1,7In animal models of autoimmune disease, sympathetic innervation or sympathectomy can alter disease severity. 8,9These findings illustrate the importance of the sympathetic nervous system in modulating immune function under normal and diseased states.
Stellate ganglion block (SGB) is a therapy for pain syndromes of the facial region and upper extremities. SGB, a partial block of cervical sympathetic nervous activity, may also affect immune activity. SGB therapy may be important for patients with chronic pain, because chronic pain can suppress immune activity. 10,11The present study was conducted to determine if SGB affects the immune response, specifically the distribution of lymphocyte subsets. In general, the percentage of total lymphocytes is classified into B, T, and natural-killer (NK) cells; furthermore, T cells are divided into inducer and helper T (CD4+) cells and suppressor and cytotoxic T (CD8+) cells. Recently, CD4+T cells have been classified functionally into helper-inducer (CD4+CD29+) cells and suppressor-inducer (CD4+-CD45RA+) cells by detecting surface antigens using monoclonal antibodies. 12Helper-inducer (CD4+CD29+) cells provide helper signals for pokeweed mitogen–induced immunogloblin synthesis by B cells. 13Suppressor-inducer (CD4+CD45RA+) cells provide poor help to B cells for pokeweed mitogen–induced immunogloblin synthesis and induce CD8+T cells, which have suppressive function. 14We therefore investigated changes of these lymphocyte subsets and NK cell activity in healthy volunteers. We also measured the plasma concentrations of catecholamines and stress hormones to investigate possible pathways involved in the mediation of the immunomodulatory effects of SGB.
Lymphocyte subsets and NK cell activity show circadian rhythms, 15and these values are subject to physical and psychological stress. 16–18Therefore, all participants were tested at the same time of day, and a crossover study was performed to determine if the injection stimulus or the drug itself affects the immune response.
Materials and Methods
Institutional and ethics committee approval was obtained, and all participants gave informed consent. Ten healthy male volunteers participated, and a crossover study was conducted. All participants received three different treatments in random order at 7-day intervals: (1) SGB with 7 ml of 1% lidocaine; (2) an identical volume of normal saline (NS) injected at the same site as SGB; and (3) intramuscular injection of 7 ml 1% lidocaine to see if lidocaine itself affects the immune response. Participants abstained from food, caffeine, and exercise for 4 h before each treatment. The treatments were performed between 9 and 11 A.M.
On the day of each treatment, participants rested in a supine position on a bed, and a 20-gauge intravenous catheter was inserted into the antecubital fossa of one arm for collection of blood samples. After catheter insertion, a cuff was placed on the opposite arm and connected to a vital-signs monitor for automated measurement of heart rate and blood pressure. After 30 min of rest, 15 ml venous blood was drawn immediately before treatment. The standard anterior paratracheal approach was used for SGB. A needle was inserted lateral to the trachea to contact the anterior surface of the C6 transverse process. Intramuscular injection was performed at the shoulder on the same side as SGB. After repeated negative aspiration tests for blood during treatment, 1% lidocaine or NS was administered over 20 s. Accuracy of the SGB was assessed by Horner’s sign on the blocked side. Participants continued to rest after 30 min of treatment, then blood samples were collected again.
Blood samples were treated with EDTA. Then 2 ml of blood was used for the analysis of lymphocyte subsets and blood cell count, and 5 ml was used for the analysis of NK cell activity. The rest of the sample was centrifuged, and the plasma was frozen at −80°C until further analysis.
Lymphocyte subsets were analyzed by flow cytometry (EPICS ELITE; Coulter, Miami, FL) using fluorescent-labeled antibodies specific to the cell markers (Coulter, Miami, FL). A 0.1-ml blood sample was incubated for 10 min with monoclonal antibodies. The samples were processed with a Q-prep Immunology Station (Coulter), which lysed in a semiautomatic fashion the erythrocytes, stabilized the leukocytes, and fixed the cells. The percentage of lymphocytes as a function of the total leukocyte count was determined by differential gating after triple-color staining. The following antibodies to lymphocyte antigens were used, and cell types were determined: CD3−CD19+(B cells), CD3+CD19−(T cells), CD3+CD4+(inducer and helper T cells), CD3+CD8+(suppressor and cytotoxic T cells), CD4+CD29+(helper-inducer T cells), CD4+CD45RA+(suppressor-inducer T cells), and CD3−CD16+CD56+(NK cells). This method for the analysis of lymphocyte subset is accurate, with a high degree of specificity and precision. In our measurements, the coefficient of variation was < 2.5%. The total leukocyte number and the percentage of total lymphocytes were measured using a cell counter (MAXM-Retic.; Coulter), and the total lymphocyte number was calculated.
Natural-killer cell activity was measured by a standard 4-h chromium release assay that was performed in 0.2-ml volumes in microplates. 19Fresh peripheral-blood mononuclear cells were isolated by gradient centrifugation of blood sample on Lymphoprep (Nycomed, Oslo, Norway). Cell viability, checked with the trypan blue exclusion method, was > 98%. Cell populations were titrated for cytolytic activity against 5,000 chromium 51-labeled K562 target cells. Effector-cell suspensions were adjusted to 1 × 106cells/ml, and an effector-to-target cell ratio of 20:1 was tested. Plates were centrifuged for 2 min at 500 ×g before incubation for 4 h at 37°C in 5% CO2, and again before collecting equal volumes of supernatant from each well. The radioactivity in the supernatant was determined using a γ counter, and the percentage of 51Cr released was calculated using the following formula:
where cpm is counts per minute. Spontaneous release was determined by incubation of target cells in medium alone, and maximum release was determined by incubation of target cells in 1N HCl. NK activity was measured in duplicate. In these experiments, we evaluated the day-to-day assay variability. The NK activity did not differ significantly on different experimental days, and the finding was replicated. In our measurements, the coefficient of variation was < 7.5%.
The plasma concentrations of epinephrine and norepinephrine were measured by high-performance liquid chromatography with electrochemical detection according to the method described by Weicker et al. 20The limit of sensitivity of this method was 1 pg/ml for each catecholamine. Commercially available radioimmunoassay kits were used for the measurement of plasma concentrations of adrenocorticotropic hormone (ACTH; Allegro HS-ACTH; Nichols Institute, San Juan Capistrano, CA) and cortisol (Amerlex Cortisol RIA Kit; Amersham, Arlington Heights, IL). The limits of sensitivity were 1 pg/ml for ACTH and 0.1 μg/dl for cortisol.
Plasma lidocaine concentrations were measured using an enzyme immunoassay method (EMIT; Syva, a Syntex Company, Palo Alto, CA) by an automatic analysis system (Aca Star; Dade International Inc., Wilmington, DE). This method for lidocaine measurement possesses a high degree of specificity and precision. In our measurements, the coefficient of variation at 0.5, 1.0, 2.5, and 5.0 μg/ml was < 10%.
Data are expressed as mean ± SD. Mean arterial pressure, heart rate, plasma concentrations of epinephrine and norepinephrine, plasma concentrations of ACTH and cortisol, the percentages of lymphocyte subsets, leukocyte number, total lymphocyte number, and NK cell activity were compared before and after treatment by the Student t test (paired). These variables were compared between groups using two-way analysis of variance followed by Duncan’s method. The plasma lidocaine concentrations after SGB and intramuscular treatments were compared using the Student t test (unpaired). Values were considered statistically significant at P < 0.05.
Participants’ age, weight, and height were 34 ± 7 yr, 62.7 ± 4.9 kg, and 170.0 ± 7.0 cm, respectively. All values before treatment were similar between the three groups. In the NS and intramuscular treatments, there was no significant difference in any value before and after treatment. After SGB, Horner’s signs were observed in all participants, but heart rate and mean arterial pressure were similar to those before SGB (table 1). After SGB treatment, the plasma concentrations of epinephrine and norepinephrine decreased significantly (table 1), but the plasma concentrations of ACTH and cortisol were unchanged. Plasma lidocaine concentrations were similar after SGB and intramuscular treatments (table 1).
Total leukocyte counts and the percentage of lymphocytes did not change significantly after SGB (table 2), but total lymphocyte counts decreased significantly (from 2,732 ± 756/μl to 2,615 ± 763/μl;P < 0.05). After SGB, significant increases were observed in the proportions of B and T cells, and that of NK cells decreased significantly (fig. 1). The proportion of each lymphocyte subset of T cells was shown relative to total T cells (100%) in fig. 2. After SGB, the proportion of CD4+cells (inducer and helper T cells) increased, and that of CD8+cells (suppressor and cytotoxic T cells) decreased, so that the CD4+/CD8+ratio increased significantly (fig. 3). The proportion of CD29+cells (helper-inducer T cells) increased significantly, but that of CD45RA+cells (suppressor-inducer T cells) did not change (fig. 2). The CD29+/CD45RA+ratio was similar before and after SGB (fig. 3).
To quantify the percent change in each lymphocyte subset after SGB, each lymphocyte subset was compared with the control value. The percent change in each lymphocyte subset is shown in figure 4. The most significant change was observed in the NK cells (−27.0 ± 5.5%). With respect to NK cell activity, significant decrease was observed after SGB (fig. 3).
After SGB, the distribution of lymphocyte subsets and NK cell activity was altered in a small but significantly different fashion. Our results also showed that the plasma concentrations of epinephrine and norepinephrine decreased after SGB. These changes were not observed with the NS and intramuscular treatments, and the plasma lidocaine concentrations after SGB and intramuscular treatments were similar. These findings indicate that the partial blockade of cervical sympathetic nervous activity by SGB modifies the distribution of lymphocyte subsets and NK cell activity, and that injection stimulus and lidocaine itself have no effect.
The precise mechanisms by which SGB alters lymphocyte subsets was not clarified in this study. It is well known that the central nervous system modulates immune activity. SBG may increase cerebral blood flow 21and subsequently may change the release of hypotha-lamic-pituitary-adrenal axis hormones. However, our results showed that the plasma concentrations of ACTH and cortisol were similar before and after SGB. This indicates that it is unlikely that the pathway that mediates the modulation of immune response by SGB is the hypothalamic-pituitary-adrenal axis.
There is evidence that the sympathetic nervous system affects the immune response. For example, in chemically sympathectomized animals, antibody and cell-mediated responses are altered. 1,7In humans, infusion of epinephrine elicits the same pattern of immune responses observed during mental stress, 22and the increase in peripheral NK cells typically observed during acute stress is inhibited by oral administration of the β-adrenoceptor antagonist propranolol. 23Furthermore, lymphocytes possess adrenergic receptors and reside in close proximity to sympathetic nerve endings in lymphatic tissue. 1Although we could not elucidate whether the effects of SGB are direct or indirect, one mechanism by which SGB alters the distribution of lymphocyte subsets may involve interaction between catecholamines and lymphocytes. Catecholamines may alter the expression of surface adhesion molecules on lymphocytes and the endothelium, thereby inhibiting migration of peripheral lymphocytes into lymphoid organs. 24Consistent with this hypothesis, Benschop et al. 25reported that catecholamines prevent the adherence of human NK lymphocytes to endothelial tissue in vivo. It has also been reported that stress or infusion of epinephrine increases NK cell activity 22and that cervical sympathectomy changes NK cell activity. 26These findings demonstrate that NK cell activity, as well as the distribution of lymphocyte subsets, is affected by sympathetic nervous activity.
The change in the percentage of NK cells was more significant than those in the other cell types. Lymphocytes have varying numbers of β2-adrenergic receptors, with NK cells having the highest. 27The high β2-adrenergic receptor number in NK cells may help to explain their greater mobilization compared with other cell types.
Neuropeptides are also candidates for immunoregulation. They are present in virtually all neurons and can be found in nerve endings at anatomic sites where they could influence immune function. 28Several neuropeptides have been identified that can mediate lymphocyte and monocyte activity by specific receptor-mediated mechanisms. 29,30For example, neuropeptide Y is colocalized with norepinephrine in sympathetic nerve terminals in the lymphoid organs. 31Although we did not measure the values of neuropeptides, it is possible that SGB could alter the outflow of these neuropeptides and may thereby change the distribution of lymphocyte subsets and NK cell activity.
Several researchers have studied the effects of stressful events on lymphocyte subsets and function. These studies reported that altered immune response follows a variety of physical stresses, including surgery, 32trauma, 33burns, 34and acute myocardial infarction. 35Physical exercise and psychological stress also modify the immune response. 16–18Consistent immunologic changes reported during stress include an increase in peripheral NK cells and suppressor and cytotoxic T cells (CD8+cells), and a decrease in the CD4+/CD8+ratio accompanied by altered NK cell activity. Our results show that SGB alters the distribution of lymphocyte subsets in a direction opposite to that reported during stress, i.e. , a decrease in CD8+and NK cells and an increase in the CD4+/CD8+ratio.
Our results show that SGB increased the CD4+/CD8+ratio. In general, an increase in the CD4+/CD8+ratio indicates that the immune system has been activated. Although helper-inducer (CD29+) T cells also increased after SGB, the helper-inducer T (CD29+)/supressor-inducer T (CD45RA+) ratio did not change, and NK cells and NK activity decreased after SGB, making it unlikely that SGB simply activates the immune system. Madden et al. 36stated that the effect of adrenoceptor stimulation on lymphocyte activity cannot be categorized as a simple inhibition or enhancement. Rather, several factors must be taken into account, including the cell types involved, the subtype of adrenoceptor stimulated, the immune stimulus, and when during the response the agonist is present.
The effects of SGB on changes in the proportion of lymphocyte subsets and NK cell activity were significant but small, and these effects were transient because the values before all treatments were similar in the crossover studies. However, our results were obtained from healthy volunteers. Further study needs to be conducted to evaluate if SGB therapy alters the immune response in patient with pain, because SGB is traditionally a therapy for pain syndromes of the facial region and upper extremities. Recent studies have shown that pain can suppress immune activity, 10,11and pain-killer drugs, including steroids and opiates, also modify the immune response. 37,38
In conclusion, a small but significant alteration in the distribution of lymphocyte subsets and NK cell activity induced by SGB indicates that local sympathetic nerve block may modulate the immune response.