Background
The induction and regulation of immune responses involve complex interactions between the immune and nervous systems mediated by the biologic action of numerous humoral factors including neurotransmitters and immunoregulatory cytokines [
1,
2]. It has been suggested that systemic somatoautonomic reflex effects following spinal manipulative therapy (SMT) might include modulation of immune reactions [
3,
4]. Animal studies have found efferent sympathetic stimulation to be immunosuppressive [
5] and it has been suggested that depressed levels of natural killer (NK) cells observed in low back patients [
6] might be related to somatovisceral reflex stimulation. However, mechanisms of SMT action on immune modulation have remained illusive [
7].
Demonstration of SMT-related effects on the production and/or biologic action of soluble regulators of the immune response provides a useful avenue for elucidating the immune consequences of SMT. Previous studies from our laboratory in asymptomatic subjects have demonstrated that a single high velocity low amplitude (HVLA) manipulation of the upper thoracic spine, characterized by cavitation and intended to mobilize a small joint fixation in the upper thoracic spine, has an inhibitory effect on proinflammatory cytokine production by peripheral blood mononuclear cells (PBMCs) [
8]. Furthermore, in the same subjects, SMT with or without cavitation caused an enhancement of the
in vitro capacity for mitogen-induced production of the immunoregulatory cytokine, interleukin-2 (IL-2) [
9].
The above observations suggested that SMT-related biological effects might indeed include a range of quantitative/qualitative changes within the integrated cytokine network. However, it is not clear if or how such changes affect the response of immune effector cells. The present study addresses this issue by investigating whether SMT-related augmentation of the
in vitro IL-2 synthesis by mitogen-activated T lymphocytes [
9] coincides with the modulation of IL-2-dependent and/or IL-2 -induced responses of normal human B cells. To this end,
in vitro antibody synthesis was determined in parallel PBMC cultures following stimulation with either pokeweed mitogen (PWM), which leads to T cell-mediated IL-2-dependent immunoglobulin (Ig) synthesis [
10] or with exogenous human recombinant IL-2 (hrIL-2), which at sufficiently high concentration induces Ig synthesis by B cells [
11].
Methods
Subjects
All subject-handling procedures were approved by the Canadian Memorial Chiropractic College Ethics Board. As indicated above, the present study represents a part of a larger investigation in which blood samples were obtained to test for changes in different parameters of the immune response following a spinal manipulative intervention [
8,
9]. In the present study, for determination of IL-2-dependent and IL-2- induced antibody production, samples were available from 74 of the subjects (Table
1).
Table 1
Demographic data of subjects.
VC ( n = 22) | 24.1 ± 1.5 | 14/8 |
SMT-NC ( n = 25) | 25.3 ± 1.21 | 15/10 |
SMT-C ( n = 27) | 24.8 ± 1.75 | 14/13 |
Details of the experimental design and protocol have been described previously [
8,
9]. Briefly, subjects were accepted into the study if they had not received any manipulative treatments in the previous 6 months and the study clinician was able to identify a restricted motion segment in the upper thoracic spine (T1-T6). Subjects in whom no restrictions could be identified were dismissed from the study. Those accepted into the study were randomly assigned to one of 3 groups: spinal manipulation with cavitation (SMT-C), spinal manipulation without cavitation (SMT-NC) or venipuncture control (VC). SMT consisted of a single high velocity low amplitude adjustment in the form of a bilateral hypothenar push (Carver Bridge) [
12], given on a single day, applied to the involved vertebral segment in a posterior-to-anterior direction, and with sufficient force, so as to produce joint cavitation as judged by the treating clinician. The procedure for SMT-NC consisted of an identical set-up using similar force but with positioning and line of drive altered to avoid cavitating the joint. In an earlier study using the same subjects, we had referred to this latter group as having received a sham manipulation [
8]. Subjects in the VC group were treated similarly to the SMT-C and SMT-NC groups in every way except for the thrust.
Blood samples
Peripheral blood was drawn in heparinized vacutainers (Becton Dickinson, Franklin Lakes, NJ) by venipuncture. Samples were collected prior to any manual intervention and then at 20 min and 2 hr post-treatment. A coding system was used in order to identify samples with a view of blinding the laboratory investigator(s) to the study groups. In every subject, samples collected before intervention served as a self-control (baseline) to which post-treatment responses were compared.
Culture system
Peripheral blood mononuclear cells (PBMCs) were separated from heparinized blood samples by fractionation on Ficoll-Paque gradients (Pharmacia Biotech, Uppsala, Sweden). Cells collected from the interface were washed three times in RPMI 1640, enumerated and suspended in complete tissue culture medium (TCM) consisting of RPMI 1640 supplemented with 10% (v/v) fetal calf serum (pre-selected for low endotoxin level), 2 mM L-glutamine, 5 × 10-5 M 2-mercaptoethanol (Sigma, St. Louis, MO) and antibiotics. To induce polyclonal antibody synthesis, duplicate PBMC cultures at a concentration of 0.5 × 106 cells/ml were stimulated, at initiation, with pokeweed mitogen (PWM, 10 μg/ml, Gibco, Grand Island, NY). Parallel preparations were stimulated with hrIL-2 derived from cDNA for human IL-2 in E. coli (Roche Diagnostics GmbH, Germany) at a final concentration of 200 U/culture according to the producer's specifications. Finally, inducer-free cultures were established in order to determine the level of spontaneous (background) synthesis of immunoglobulins (Igs) in each subject. All preparations were cultivated for 7 days in a humidified atmosphere of 5% CO2 and 95% air. At the end of incubation period, the culture supernatants were collected, aliquoted and were stored at -78°C. Samples were thawed immediately before testing and, to minimize inter-assay variability, all culture supernatants derived from a given subject were always examined in the same experiment.
Phenotypic analyses of PBMCs
Enumeration of peripheral blood B and T lymphocytes in the preparations of PBMCs collected at baseline and then 2 hr post-treatment was carried out by flow cytometric analysis of samples following immunofluorescent staining with the respective anti-CD19 and anti-CD3 mouse anti-human monoclonal antibodies (BD Biosciences, Mississauga, ON).
Assessment of immunoglobulin production
Polyclonal Ig synthesis was determined using the enzyme-linked immunosorbant assay (ELISA) technique essentially as described previously [
13]. Briefly, duplicate dilutions of standards or culture supernatants in PBS-Tween were added to flat bottom microplate wells (Immulon 2HB, Thermo Labsystem, Franklin, MA) coated with anti-human immunoglobulin G (IgG) or immunoglobulin M (IgM) and incubated for 2 hr at 37°C. The plates were then washed thoroughly in PBS-Tween and incubated again (1 hr, 37°C) with a predetermined concentration of peroxidase-conjugated goat anti-human IgG or IgM. Following the development of color in the presence of a 0.4% solution of orthophenylenediamine (Sigma, ST. Lois, MO) and H
2O
2, the absorbance was measured at 492 nm using a Titertek Multiscan (Flow Laboratories, Helsinki, Finland). Concentrations of a given Ig were calculated from linearized (best fit) standard curves. Detection level for both IgG and IgM was 30 ng/ml. Each culture supernatant was tested at least 3 times and at several dilutions.
Statistics
Levels of the induced IgG and IgM production in the study groups were evaluated for normality using the Shapiro-Francia test, and for equality of variances between groups using Levene's robust test statistic. For both outcome measures data were determined to be non-normal and equality of variances was not confirmed. As a result of these findings, the data was transformed prior to completing further analysis. The IgG data were transformed using the Box-Cox method, and thereafter were found to be normally distributed with equal variances between groups. The IgM data were transformed by taking the natural log of each value, and thereafter were also found to be normally distributed with equal variances between groups. Statistical significance of differences between the study groups (V C, SMT-NC, SMT-C) and within groups (baseline vs. post-treatment at 20 min vs. post-treatment at 2 h) was then determined using the repeated measures ANOVA. This was followed by post-hoc Tukey's HSD test for pairwise comparisons at each time point [
14]. Statistical significance was accepted at p < 0.05. Data were analyzed using STATA SE 8 Sofware.
Discussion
Results of the present investigation demonstrate that in normal asymptomatic subjects in whom a restricted upper thoracic motion segment was identified, neither venipuncture alone nor a single spinal manipulation with or without cavitation affected the capacity for the IL-2 -dependent (i.e. T-cell-dependent), PWM-triggered antibody production examined within 2 hr post-intervention. However, within the same time frame, antibody synthesis (both IgG and IgM class) induced by hrIL-2 was significantly augmented in cultures from subjects treated with SMT-C.
The mechanism(s) underlying the significant amplification of the response to exogenous IL-2 in SMT-C treated subjects is unknown. The possibility that the observed effect was related to an increase in the total content of IL-2 in these cultures cannot be excluded. The IL-2-inducible immunoglobulin synthesis is a dose-dependent process and requires high concentrations of this cytokine [
16]. As reported previously, the intrinsic capacity for IL-2 production in cultures from SMT-treated subjects is enhanced [
9]. Considering the fact that IL-2 up-regulates its own production, as well as the expression of specific IL-2 receptors [
17,
18], it is feasible that the production of endogenous IL-2 was indeed up-regulated in the presence of hrIL-2, and more so in subjects treated with SMT-C. Furthermore, the increase in the total IL-2 level could facilitate the release of other soluble mediators of the humoral immune response by functional T cells present in the studied cultures and subsequently enhance antibody secretion by B cells [
19]. Noteworthy, a significant increase in the level of IgG production was observed also, at 2 hr post-treatment, in subjects who received SMT-NC manipulation (Figure
2A). This is consistent with our earlier findings of the time-limited effect of SMT-NC on T lymphocytes [
9].
The above considerations notwithstanding, it is doubtful that the combined action of endogenous and exogenous IL-2 could be the sole mechanism of the observed up-regulation of IL-2-induced Ig synthesis in the SMT-C group. Normal human B cells express functional (high affinity) IL-2 receptors (IL-2R) and thus IL-2 plays a significant role in the modulation of B cell function [
20]. Therefore, it is feasible that following SMT-C, the interaction between IL-2 and its specific high affinity receptor (IL-2R) on the surface of B lymphocytes was somewhat facilitated and resulted in augmentation of Ig synthesis. However, the effect of SMT-C on the capacity of B lymphocytes for the expression of IL-2R was not investigated in this study.
It is also possible that the increase in IL-2 induced antibody production in SMT-C treated subjects was related, directly or indirectly, to the biologic action(s) of other soluble mediators released as a consequence of spinal manipulation. The cross- talk between the soluble mediators produced by the immune and nervous systems regulates the magnitude and duration of both immune and inflammatory responses [
21,
22]. Indeed, the observation of attenuated production of proinflammatory cytokines in subjects treated with SMT-C [
8] prompted our exploratory studies on potential mechanisms of this effect. Studies still in progress in this laboratory indicate that PWM-activated cultures from SMT-C -treated, but not SMT-NC or VC subjects contain significantly elevated levels of the anti-inflammatory cytokine interleukin 10 (IL-10) [
23]. IL- 10 has been shown to increase the affinity of the B cell receptor for IL-2 resulting in a putative improvement of signal transduction and promotion of B lymphocyte activation [
24]. Furthermore, IL-10 synergizes with the available IL-2 to increase synthesis of Igs but has no effect on T-cell dependent polyclonal responses [
25‐
27]. In the present study PWM-induced, T-cell dependent antibody synthesis was indeed not altered following SMT (Figure
1). Thus, it is feasible that IL-2-induced IgG and IgM production, in cultures obtained from SMT-C treated subjects (Figure
2), was augmented due to enhancement of IL-2 signalling by endogenous IL-10.
The suggested facilitation of Ig synthesis due to SMT may be associated with joint cavitation. However, in this regard the design of our experiments did not control or measure the actual forces delivered during the manipulative procedure. Although the intention was to deliver a manipulative thrust of similar force (but different direction) for both the cavitation and no cavitation groups, the forces delivered to the no cavitation group may have been smaller. We have previously discussed the issue of cavitation in the context of the effects of manipulation [
9].
The clinical significance of the elevated responsiveness to IL-2 demonstrated in this
in vitro study is presently unclear. It should be noted that augmentation of IL-2-induced IgG or IgM synthesis in the SMT-C group, although statistically significant, did not exceed the physiological range of normal human response [
13,
28]. Nonetheless, results of the present pilot study provide the first experimental evidence that systemic sequelae of spinal manipulative therapy include functional changes in the ability of peripheral blood lymphocytes to respond to immunoregulatory mediators and the clinical relevance of such alterations should be further explored.
List of Acronyms
ELISA - Enzyme linked immunosorbant assay, hrIL-2 - Human recombinant interleukin 2, HVLA - High velocity low amplitude, Ig - Immunoglobulin, IgG - Immunoglobulin G, IgM - Immunoglobulin M, IL-2 - Interleukin 2, IL-2R - Interleukin 2 receptor, IL-10 - Interleukin 10, PBMC - Peripheral blood mononuclear cell, PBS - Phosphate buffered saline, SMT - Spinal manipulative treatment (or therapy), SMT-C - Spinal manipulative treatment associated with cavitation, SMT-NC - Spinal manipulative treatment without cavitation, VC - Venipuncture control
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JTI contributed to the design of the study, was responsible for all laboratory procedures, analysis of data, and contributed to the writing of the manuscript. MM performed statistical analysis and contributed to writing of the manuscript. HSI contributed to the design of the study, was responsible for subject recruitment and coordination of the study, analysis of data, and contributed to the writing of the manuscript. RR contributed to the design of the study and was the study clinician. All authors have read and approved the final manuscript.