We have previously reported that hot-spring hydrotherapy for a short duration can change hormone levels in the blood and quantitatively as well as qualitatively regulate leukocytes and their subpopulations. In this study, we examined the effect of hot-spring hydrotherapy on the lymphocytes expressing β₂-adrenergic receptors (β₂-ARs). We obtained peripheral blood from 9 healthy volunteers (age, 49.7±11.2 years) at the same sampling time before and after (the next day) hot-spring hydrotherapy, and measured the total and differential leukocyte counts and the levels of 3 catecholamines (adrenaline, noradrenaline, and dopamine). We also analyzed the β₂-AR of lymphocyte subsets by combination with fluorescent monoclonal antibodies (CD3, CD4, CD 8, CD 19, and CD56) by using flow cytometry.
 The number of granulocytes significantly decreased after hot-spring hydrotherapy (p < 0.05). The ratio of β₂-AR⁺ CD4⁺ cells and β₂-AR⁺ CD3⁺ cells was low, but that of β₂-AR⁺ CD56⁺ cells and β₂-AR⁺ CD8⁺ cells was high. The CD8⁺ cell and CD56⁺ cell counts tended to increase after hot-spring hydrotherapy, but no significant variation was found in the β₂-AR⁺ cells of each subset. Adrenaline levels also significantly decreased after hot-spring hydrotherapy (p < 0.05). The rate of change in adrenaline levels before and after hot-spring hydrotherapy was highly correlated with the rate of change in the levels of CD56⁺, CD8⁺, and β₂-AR⁺ CD56⁺ cells. Thus, we found a difference in thepercentage of β₂-AR-expressing cells in the lymphocyte subsets. Therefore, it is possible that a short duration of hot-spring hydrotherapy decreased adrenaline levels and quantitatively influenced the natural killer cells and CD8⁺ cells, which express large amounts of β₂-AR.

In order to investigate an effects of the hot spring within a short period on immune system of human, the leukocyte, monocytes, lymphocyte and lymphocyte surface markers: CD2, CD4, CD8, CD16, CD19 and CD57 were tested in the human peripheral blood of twenty-three healthy volunteers by hot spring bathing. The results were as follows: Total number of leukocytes and lymphocytes in the peripheral blood significantly increased in an older group after hot spring bathing (p<0.01). However, we obtained a clear decrease in the number of granulocyte after hot spring bathing in the younger group (p<0.01). In addition, we found greater increase of the CD16⁺ cell counts and a clear decrease of the CD19⁺ cell counts in older group. But in younger group, we also obtained an increase of CD8⁺, CD16⁺ cells after hot spring bathing. These results indicated that hot spring bathing can regulate the physical immune system.
According to the percentage of lymphocytes or granulocytes in the total leukocytes, volunteers were divided into two types, more than 70% of granulocyte were recognized as G type and more than 40% of lymphocyte were divided in the L type. We found an increase of lymphocyte and lymphocyte subsets as well as a decrease in granulocyte in G group by hot spring bathing. But in L group, especially, indicated a greater increase in granulocyte and a decrease in lymphocyte subsets. We suggest that hot sping bathing can regulate by an autonomic nerve system, making it suitable.

We have simultaneously proved that cell populations taking charge of immunity in human peripheral blood can be regulated quantitatively by hot spring bathing. Now, we investigated the effect of hot spring bathing qualitatively on cytokine production by lymphocyte cell in human peripheral blood estimating by cytokine containing cell by FACScan. We found a significant increase in IFN-γ containing cells after hot spring bathing and an increase in IL-4 with no statistical significance after hot spring bathing. In addition, we found significant negative relationship between the level of IFN-γ, IL-4 and IL-1β before hot spring bathing and the ratio of cytokine that increased in variation after hot spring bathing. Namely, after hot spring bathing, there was a decrease of cytokine producing cells in subjects who had higher level before hot spring bathing. But an increase in subjects who had lower level before hot spring bathing, the trend was concentrated toward average levels in the cytokine production by lymphocyte in peripheral blood. So we suggest that hot spring bathing can promote acquired immunity to make it possible more suitable as immune reaction.