Osteopathic manipulative therapy induces early plasma cytokine release and mobilization of a population of blood dendritic cells

Abstract

It has been claimed that osteopathic manipulative treatment (OMT) is able to enhance the immune response of individuals. In particular, it has been reported that OMT has the capability to increase antibody titers, enhance the efficacy of vaccination, and upregulate the numbers of circulating leukocytes. Recently, it has been shown in human patients suffering chronic low back pain, that OMT is able to modify the levels of cytokines such as IL-6 and TNF-α in blood upon repeated treatment. Further, experimental animal models show that lymphatic pump techniques can induce a transient increase of cytokines in the lymphatic circulation. Taking into account all these data, we decided to investigate in healthy individuals the capacity of OMT to induce a rapid modification of the levels of cytokines and leukocytes in circulation. Human volunteers were subjected to a mixture of lymphatic and thoracic OMT, and shortly after the levels of several cytokines were evaluated by protein array technology and ELISA multiplex analysis, while the profile and activation status of circulating leukocytes was extensively evaluated by multicolor flow cytometry. In addition, the levels of nitric oxide and C-reactive protein (CRP) in plasma were determined. In this study, our results show that OMT was not able to induce a rapid modification in the levels of plasma nitrites or CRP or in the proportion or activation status of central memory, effector memory or naïve CD4 and CD8 T cells. A significant decrease in the proportion of a subpopulation of blood dendritic cells was detected in OMT patients. Significant differences were also detected in the levels of immune molecules such as IL-8, MCP-1, MIP-1α and most notably, G-CSF. Thus, OMT is able to induce a rapid change in the immunological profile of particular circulating cytokines and leukocytes.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Levels of nitrites and CRP in plasma of OMT and sham experimental groups at 5 and 30-treatment.

Plasma was recovered from blood samples and analyzed for the presence of nitrites (A) and CRP (B) as described in the Materials and Methods section. Data are expressed as mean ± SD. Data were analyzed by ANOVA followed by Tukey-Kramer Multiple Comparisons post-test. N = 10 OMT and 10 sham.

Figure 2. Total and differential WBC counts in OMT and sham experimental groups at 5 and 30-treatment.

The total and differential numbers of white blood cells were determined by determined in an automatic cell counter in triplicate (HemaVet HV950FS programmed for human blood). Data are expressed as mean ± SD. Data were analyzed by ANOVA followed by Tukey-Kramer Multiple Comparisons post-test. N = 10 OMT and 10 sham.

Figure 3. Flow cytometry analysis of circulating NK and CD3 T cell populations in OMT and sham experimental groups at 30-treatment.

PBMC were obtained from blood samples and stained for CD45, NK and CD3 with fluorescent antibodies and analyzed by multicolor flow cytometer within the lymphocyte FSC vs. SSC gate. The percentage of NK and CD3 cells within the CD45 population was determined. Data are expressed as mean ± SD. Data were analyzed by ANOVA followed by Tukey-Kramer Multiple Comparisons post-test. N = 10 OMT and 10 sham.

Figure 4. Levels of nitrites and CRP in plasma of OMT and sham experimental groups at 30 and 60-treatment.

Plasma was recovered from blood samples and analyzed for the presence of nitrites (A) and CRP (B) as described in the Materials and Methods section. Data are expressed as mean ± SD. Data were analyzed by ANOVA followed by Tukey-Kramer Multiple Comparisons post-test. N = 16 OMT and 17 sham.

Figure 5. Total and differential WBC counts in OMT and sham experimental groups at 30 and 60-treatment.

The total and differential numbers of white blood cells were determined in an automatic cell counter in triplicate (HemaVet HV950FS programmed for human blood). Data are expressed as mean ± SD. Data were analyzed by ANOVA followed by Tukey-Kramer Multiple Comparisons post-test. N = 16 OMT and 17 sham.

Figure 6. Flow cytometry analysis of circulating NK, NKT and T cell populations in OMT and sham experimental groups at 60-treatment.

(A) Gating strategy to define NK, NKT and T cell percentages within the CD45 fraction of PBMC. (B) Percentage of CD3, NK and NKT cells within the CD45 PBMC fraction. Distribution of different CD4 (B) and CD8 (C) populations within the CD3/CD45 fraction of PBMCs. Data are expressed as mean ± SD. Data were analyzed by ANOVA followed by Tukey-Kramer Multiple Comparisons post-test. N = 16 OMT and 17 sham.

Figure 7. Activation status of circulating NK, NKT and T cell populations in OMT and sham experimental groups at 60-treatment.

Mean fluorescence intensity (MFI) values of CD69, an early activation antigen, were determined in NK and NKT cells (A), and Central and effector memory and naïve CD (B) and CD8 (C) T cells. Data are expressed as mean ± SD. Data were analyzed by ANOVA followed by Tukey-Kramer Multiple Comparisons post-test. N = 16 OMT and 17 sham.

Figure 8. Flow cytometry analysis of circulating APC populations in OMT and sham experimental groups at 60-treatment.

B cells and different subpopulations of monocytes and dendritic cells were analyzed within the HLA-DR fraction of PBMCs. (A) Gating strategy to define antigen presenting cells. (B) Percentage of B cells, monocytes and DCs within the HLA-DR fraction. Distribution of different monocyte (C) and dendritic cell (D) populations within the APC fraction of the PBMCs. Percentages of the different populations were determined by comparison with isotype controls Data are expressed as mean ± SD. Data were analyzed by ANOVA followed by Tukey-Kramer Multiple Comparisons post-test. N = 16 OMT and 17 sham.

Figure 9. Activation status of circulating APC populations in OMT and sham experimental groups at 60-treatment.

MFI values of CD80, CD86 and HLA-DR were determined in B cells (A) and plasmacytoid DCs (B) present in the PBMC fraction of OMT and sham-treated groups 1 h after treatment. MFI values of CD80 and HLA-DR were determined in monocyte (C) and dendritic cell subpopulations (D) present in the PBMC fraction of OMT and sham-treated groups 1 h after treatment. Data are expressed as mean ± SD. Data were analyzed by ANOVA followed by Tukey-Kramer Multiple Comparisons post-test. N = 16 OMT and 17 sham.

Figure 10. Multiplex analysis of plasma chemokine and cytokine populations in OMT and sham experimental groups at 30 and 60-treatment.

The levels of specific chemokines, cytokines or growth factors were determined by using a custom Milliplex Map Kit human cytokine panel. Data are expressed as mean ± SD. Data were analyzed by ANOVA followed by Tukey-Kramer Multiple Comparisons post-test. When samples did not follow normal distribution, non-parametric statistics were used for analysis. When comparing the levels of a particular molecule within the OMT or sham group at different time-points, we used paired-sample statistics analysis. Overall, the statistical analysis was focused on each specific cytokine expressed by supernatants recovered from different environments, but no comparison was performed between different cytokines. N = 12 OMT and 14 sham.