Effects of Lactobacillus spp. on Cytokine Production by RAW 264.7 Macrophage and EL-4 Thymoma Cell Lines

We have hypothesized that lactobacilli used in fermented dairy products can stimulate immune function via enhancing cytokine secretion by leukocytes. To test the effects of lactobacilli on cytokine production, RAW 264.7 cells (macrophage model) and EL4.IL-2 thymoma cells (T helper cell model) were cultured in the presence of 16 representative strains of heat-killed Lactobacillus spp. cells. In unstimulated RAW 264.7 cells, most lactobacilli, when present at concentrations from 10⁶ to 10⁸ bacterial cells per ml, induced marked tumor necrosis factor alpha (TNF-α) production (up to 411-fold) compared to the negligible TNF-α in controls. A strain-dependent increase in interleukin 6 (IL-6) production (up to 88-fold) was also observed without stimulation at concentrations of 10⁸ bacteria per ml. Upon concurrent stimulation of RAW 264.7 cells with lipopolysaccharide, both IL-6 and TNF-α production were enhanced between 4.2- and 10.6-fold and 1.8- and 8.7-fold, respectively, when cultured with 10⁸ lactobacilli per ml. In unstimulated EL4.IL-2 cells, lactobacilli had no effect on interleukin 2 (IL-2) or interleukin 5 (IL-5) production. Upon stimulation of EL4.IL-2 cells with phorbol 12-myristate-13-acetate, IL-2 secretion increased up to 1.9-fold in the presence of 10⁶ L. bulgaricus Lr 79 cells per ml whereas this cytokine decreased in the presence of 10⁷ or 10⁸ lactobacilli per ml. In contrast, IL-5 secretion increased in the presence of increasing concentrations of lactobacilli (up to 3.1-fold with 10⁸ L. bulgaricus NCK 231 cells per ml). The results indicated that direct interaction of most lactobacilli with macrophages resulted in a concentration-dependent enhancement of cytokine production, whereas the effects on cytokine production by the T-cell model were smaller and strain dependent. The in vitro approaches employed here should be useful in further characterization of the effects of lactobacilli on the gut and systemic immune systems.