Korean J Physiol Pharmacol.  2012 Aug;16(4):225-230. 10.4196/kjpp.2012.16.4.225.

Immunostimulatory Effects of beta-glucan Purified from Paenibacillus polymyxa JB115 on Mouse Splenocytes

Affiliations
  • 1Laboratory of Veterinary Pharmacology, College of Veterinary Medicine, Jeju National University, Jeju 690-756, Korea. jooh@jejunu.ac.kr

Abstract

We investigated the effects of beta-glucan purified from Paenibacillus polymyxa JB115 on the viability and proliferation of splenocytes. Splenocytes play a critical role in host immunity. MTT assays and trypan blue exclusion tests revealed that beta-glucan significantly promoted the viability and proliferation of splenocytes over a range of concentrations. However, there was no specific subset change. beta-glucan protected splenocytes from cytokine withdrawal-induced spontaneous cell death. For further mechanistic studies, ELISA assay revealed that beta-glucan enhanced the expression of anti-apoptotic molecules and interleukin 7 (IL-7), a cytokine critical for lymphocyte survival. We also investigated the IL-2 dependency of beta-glucan-treated splenocytes to determine if treated cells could still undergo clonal expansion. In flow cytometric analysis, beta-glucan induced increased levels of the activation marker CD25 on the surface of splenocytes and beta-glucan-treated splenocytes showed higher proliferation rates in response to IL-2 treatment. This study demonstrates that beta-glucan can enhance the survival of splenocytes and provides valuable information to broaden the use of beta-glucan in research fields.

Keyword

beta-glucan; Anti-apoptosis; IL-2 dependency; Splenocytes; Viability

MeSH Terms

Animals
Cell Death
Dependency (Psychology)
Diminazene
Enzyme-Linked Immunosorbent Assay
Interleukin-2
Interleukin-7
Lymphocytes
Mice
Paenibacillus
Plasmodiophorida
Trypan Blue
Diminazene
Interleukin-2
Interleukin-7
Trypan Blue

Figure

  • Fig. 1 The enhanced viability and proliferation of splenocytes by β-glucan. The splenocytes were cultured at a concentration of 2×105 cells/200 µl in 96-well culture plates. (A) Splenocytes were treated with 0~200 µg/ml of β-glucan for 3 days and MTT assays were performed. (B) Splenocytes were treated with 0~100 µg/ml of β-glucan for 3 days and trypan blue exclusion tests were performed to count viable/dead cells. *,**,***indicate p<0.05, 0.01, and 0.001, respectively, as compared with the control. Data are mean±SD from three or four individual wells.

  • Fig. 2 β-glucan treatment does not alter the proportion of splenic subsets. Splenocytes were treated with 0, 20, or 100 µg/ml for 2 days. The cells were stained for CD4, CD8, and CD19, and analyzed using a flow cytometer. The number in the histograms indicates the percentage of a specific subset. A representative data set is shown from three independent experiments with similar results.

  • Fig. 3 β-glucan protects splenocytes against spontaneous cell death. Splenocytes were cultured at a concentration of 5×106 cells/5 ml in 6-well culture plates with 0, 20, or 100 µg/ml β-glucan treatment for 2 days. (A) The cell size (FSC/SSC) analysis, (B) annexin V-FITC/PI staining, and (C) Rhodamine 123 staining were performed by a flow cytometer. The number indicates cell percentage (A), the cell percentages in quadrants (B), and the cell percentage with high MFI (C), respectively. Each flow cytometric data set is presented from three independent experiments with similar results.

  • Fig. 4 The altered expression of apoptosis-related molecules in β-glucan-treated splenocytes. Splenocytes were treated as described in Fig. 3 and the cell lysates were prepared for Western blot analysis. A representative image set was selected from two independent experiments with similar results (A). After measuring band densities, the ratio of each band to beta-actin was calculated (B).

  • Fig. 5 β-glucan induces the production of IL-7 in splenocytes. Cells were treated in 96-well plates and their supernatants were used for ELISA analysis to determine IL-2 (A), IL-7 (B) concentrations. **indicates p<0.01 compared to control. Data presented is from three independent experiments with similar results.

  • Fig. 6 β-glucan increases spleen cell sensitivity to IL-2. Splenocytes were treated as described in the Methods. Cells were stained for CD25, CD69, or CD45RB (A) and examined using a lymphocyte proliferation assay with CFSE to check IL-2 dependency (B, C). The number indicates the percentage of cells with high MFI (A), the percentage of smaller/bigger cells in treated splenocytes responding to IL-2 (B), and cells with low MFI (C), respectively. A representative data set was selected from three independent experiments with similar results.


Cited by  1 articles

Evaluation of adjuvant effects of fucoidan for improving vaccine efficacy
Su-Yeon Kim, Hong-Gu Joo
J Vet Sci. 2015;16(2):145-150.    doi: 10.4142/jvs.2015.16.2.145.


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