Two of the most intriguing controversies surrounding the chiropractic profession are: 1) the biological mechanisms associated with spinal manipulation 12, and 2) the clinical impact of the audible release that comes from a high velocity, low amplitude force directed to a spinal joint 3. The research reported here adds to the body of knowledge in each of these important areas: samples of cell culture supernatants obtained in the course of an earlier study examining the effect of spinal manipulation on selected parameters of the immune response 4 were evaluated for changes in the capacity for production of the immunoregulatory cytokine, interleukin 2 (IL-2), pre- and post-intervention, as well as, between manipulation characterized by an audible release (cavitation) and manipulation without audible release.

The current model of spinovisceral (somatoautonomic) reflex provides a link between spinal manipulation, the autonomic nervous system (ANS) and various visceral functions 5. Although experimental studies have demonstrated that somatoautonomic reflex effects are evident in various non-musculoskeletal systemic reactions 6 studies in humans are still limited, particularly with respect to the effect of spinal manipulative therapy (SMT) on the integrated responses of the nervous and immune systems. Clarification of such a relationship can have clinical implications in the area of manual interventions in pursuit of somato-visceral outcomes. Our recent studies on asymptomatic subjects receiving a single high velocity low amplitude spinal manipulation (HVLA), characterized by cavitation (audible release), in the thoracic spine, demonstrated suppression of the in vitro production of two proinflammatory cytokines, tumor necrosis factor α (TNFα) and interleukin 1β (IL-1β) 4 relative to subjects receiving a high velocity, low amplitude force applied to the thoracic spine, without cavitation. Earlier studies by Brennan et al. 7 suggested that a series of high force, HVLA manipulations of the lumbosacral spine of low back patients, as well as asymptomatic controls, did not result in quantitative changes of lymphocyte subpopulations in peripheral blood samples collected from either group. To our knowledge, however, the effect of SMT (of any type) on the functional activity of these cells, including their capacity for cytokine production, has not been studied.

IL-2, originally identified as a T-cell growth factor, is now recognized as a pivotal cytokine in T-cell dependent immune responses 8. An increasing body of evidence indicates also that IL-2 plays a major role in the development, maintenance and survival of regulatory T cells warranting its critical importance in the induction and sustenance of immune tolerance 9. Determinations of in vivo (serum) IL-2 levels have been applied in clinical studies in which systemic activation of T-cells is suspected 10. On the other hand, assessment of the in vitro production of this cytokine by mitogen-stimulated human cell cultures has been long used as a standard procedure for scrutinizing the capacity of T lymphocytes to become activated 11. Thus, both in vivo and in vitro determinations of IL-2 have been utilized in this investigation. Furthermore, the in vitro induction of IL-2 synthesis may be achieved either via mitogen-related transmembrane signaling or by a chemical stimulation which bypasses such requirements. Both methods have been utilized in the present study.

It has been suggested that cavitation (the audible release) associated with HVLA is what "truly" distinguishes SMT from other manipulation-type modalities 12. The intervention methods used in this investigation are consistent with this notion. That is, HVLA forces were applied to some subjects with the intent of producing an audible release, and in other subjects with the intent of not producing a release. Controversy remains as to whether cavitation results in any clinically relevant differences 1314. Studies have not yet been conducted to determine if differences due to cavitation exist at a cellular/molecular or immunological level. This investigation begins such an exploration.

The biological mechanisms associated with spinal manipulation are poorly understood 1. However, evidence has been accumulating that somatovisceral effects may result from a spinal manipulative therapy 6. We have previously demonstrated that a single spinal HVLA manipulation, characterized by cavitation and intended to mobilize a small joint "fixation" in the upper thoracic spine in asymptomatic subjects, has a suppressing effect on proinflammatory cytokine synthesis by PBMCs in vitro 4. Earlier studies have demonstrated increased activity of the innate immune response components following a single SMT in the upper T-spine of asymptomatic subjects 2223. However, parameters of specific immunity have not been investigated with a view of determining possible alterations in lymphocyte functions. In the present study we have demonstrated that following a spinal manipulative intervention the in vitro synthesis of IL-2 increases significantly, both with and without cavitation, for at least the short term (20 min post-SMT). These results are important from two different perspectives: a) the effect (increase) may be independent of joint cavitation and b) the response is evident in mitogen (SPA)-activated cultures (Figs 1A, 1B) but not those stimulated by the combination of TPA and calcium ionophore (Figs 2A, 2B)

The design of our study does not allow for speculation as to the mechanism(s) involved in the transduction of a putative effect of the manipulative thrust to the cellular/molecular level in vivo. However, the in vitro application of two inducers dissimilar with respect to the route by which the activating signal was provided allows for an insight into the mechanism of SMT action in our experimental model system. SPA is suggested to induce preferential activation of CD4+ (Th1) cells through its ability for cross-linking with the MHC class II molecules on the surface of antigen presenting cells APC 24. The application of phorbol ester and calcium ionophore by-passes this requirement and mobilizes molecular mechanisms of IL-2 synthesis directly 2526. The results of the present study demonstrate clearly that manipulative intervention provided by spinal manipulation of the thoracic spine, results in a significant augmentation of IL-2 secretion in vitro induced by SPA.

The increased IL-2 synthesis by PBMCs was not accompanied by a systemic (in vivo) release of IL-2 in response to any of the interventions. Thus, at baseline, serum IL-2 was detected in a small number of subjects and did not increase post-manipulation. Similarly, constitutive (spontaneous) in vitro secretion of IL-2 remained consistently comparable in all study groups. It is therefore unlikely that the observed increments in SPA-induced production of IL-2 ensued from in vivo (systemic) pre-activation of circulating peripheral blood T lymphocytes. Rather, it is possible that the manipulative interventions had a priming effect on cells/factors in vivo with a subsequent enhancing effect on the interaction between antigen presenting cells, principally dendritic cells (DCs), and T lymphocytes. Such improved interaction has been shown to be mediated by another cytokine, interleukin 15 (IL-15) resulting in increased IL-2 production by T lymphocytes, such as observed in our study. Interestingly, IL-15 which is highly expressed in neural tissues and skeletal muscles 27 is released in large quantities following physical stress 2829.

In the present study, it is not known if any of the procedures (including the "laying on of the hands", the manipulative thrust) to which participants were subjected to, were sufficiently stressful to cause an increase in the systemic output of IL-15. Venipuncture controls were examined and touched by the clinician in a manner comparable to the SMT groups, making it unlikely that the observed effect on IL-2 synthesis is due to "laying on of the hands" or anticipation-related stress. What remains common to both SMT-C and SMT-NC is the manipulative force. It has been reported that a threshold force may be required in order to attain SMT-related physiological changes 23. Manipulative forces were not measured in this study and we cannot stipulate as to the magnitude of force to which participants in the SMT-NC or SMT-C groups were subjected. However, it is feasible that manipulative forces and intervention-related stress increased the systemic levels of IL-15 in both SMT-C and SMT-NC groups. This could improve DC interaction with T lymphocytes and increase IL-2 production. Finally, IL-15-activated DCs themselves produce IL-2 in considerable quantities 30. The process of DC stimulation resulting in IL-2 synthesis requires the presence of both T lymphocytes and microbial product. 31. In the present study, these conditions were met in SPA- but not TPA-stimulated cultures of PBMCs, which overall, showed no alterations in IL-2 production (Figs 2A, 2B).

The observed IL-2 increase might also be due to an increase in the number of DCs. Earlier studies have revealed no quantitative changes in T cells; however, enumeration of PBMCs expressing the DC phenotype was not done. It has been demonstrated that physical stress increases the number of DC cells in the circulation 32. Thus, an increase in the number of IL-2 secreting DCs in the PBMC preparations examined after the intervention, might explain the observed increase in IL-2 production in cultures derived from SMT-C and SMT-NC – treated subjects. Future studies of possible alterations in DC numbers and biological responses following manipulative interventions should yield informative results in this regard.

The results of this study indicate that the increase in IL-2 synthesis by PBMCs due to a manipulative intervention was independent of joint cavitation when considered in the 20 minute time frame following SMT. Statistical analysis of the data for 2 hours post intervention revealed similar results. However, in the absence of cavitation there was an attenuation of the effect (Fig 1B) suggesting that joint cavitation may have a longer lasting effect. It may be argued that increasing the number of participants in the study groups might have delineated differences between the SMT-C and SMT-NC groups more clearly. However, the p values (0.006 or less) revealed by the statistical analysis indicate that the study had adequate power. Nevertheless, future studies will benefit from reproducing these results with similar or better statistical power, and more importantly, exploring the time course of IL-2 synthesis, by making determinations over a longer period (e.g. 1, 2, 3, and 24 hours) following SMT.

While there is controversy around the topic of the clinical significance of cavitation 131433 the results reported here, as well as earlier findings of cavitation-dependent-suppression of proinflammatory cytokines in response to SMT 4 suggest, that depending on the time-frame of determinations and/or type of outcomes investigated, cavitation may be an important qualifier of a successful HVLA thrust. A clear association between manipulation of the lumbar spine and zygapophyseal joint gapping has been demonstrated by use of magnetic resonance imaging in normal subjects 34. However, a relationship between joint gapping and cavitation remains to be established. It has been suggested that while cavitation may be a reliable indicator of joint gapping, the mechanical effects of spinal manipulation may not be directly related to cavitation 21233. Flynn et al. 14 found that an "audible pop" did not change the outcome for sacroiliac region manipulation in patients with non-radicular low back pain. In their study all participants were manipulated similarly and were grouped into "audible pop" and "no audible pop" post manipulation. However, in this type of design it is possible that the perceived absence of an "audible pop" is due the fact that articular release may occur without being audible 35. In the present study the SMT-NC group received a manipulative thrust designed not to produce cavitation. Nevertheless, we realize that in order to make an assertion with respect to the occurrence or absence of cavitation more rigorous means need to be employed. To this end the use of accelometry 36 in future studies will be useful. In addition, quantification of manipulative forces will shed further light on the relationship between IL-2 synthesis and HVLA with or without cavitation.

A single HVLA manipulation to the thoracic spine of asymptomatic subjects causes a significant enhancement in IL-2 secretion by PBMCs in vitro. This increase is independent of joint cavitation at least for the short term. However, the duration of the effect appears to be time-restricted as evidenced by the decline of IL-2 levels 2 hours post-intervention in the non-cavitating group. These observations suggest that under certain physiological conditions, SMT could influence, directly or indirectly, IL-2-regulated biological responses. The results provide impetus for further investigations in order to clarify the relationship between HVLA manipulation with or without cavitation and the synthesis of IL-2 and other immunoregulatory mediators.

The authors declare that they have no competing interests.

JTA contributed to the design of the study, was responsible for all laboratory procedures, analysis of data, and contributed to the 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, MM performed the statistical analysis of the data and contributed to the writing of the manuscript, GMH contributed to data analysis and 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 version of the manuscript.