Differential Response of B Cells to an Immunogen, a Mitogen and a Chemical Carcinogen in a Mouse Model System

Abstract

Background: B cells are specific antibody generating cells which respond to foreign intruders in the circulation. The purpose of this study was to compare the relative immunogenic potentials of three well established agent types viz. an immunogen, a mitogen and a carcinogen, by following B cell responses to their presence in a mouse model system. Methods: Mice were treated with tetanus toxoid (immunogen), poke weed mitogen (typical mitogen), and benzo-α- pyrene (carcinogen) and generated B cell populations were determined in isolated splenic lymphocytes (splenocytes) by flow cytometry using specific anti-B cell marker antibodies. Flow cytometric estimation of LDL receptor (LDLR) expression, along with associated B cell markers, was also conducted. Kit based estimation of serum IgG, western blotting for LDLR estimation on total splenocytes and spectrometry for cholesterol and serum protein estimation were further undertaken. Student’s T-tests and one way ANOVA followed by the Bonferroni method were employed for statistical analysis. Results: The mitogen was found to better stimulate B cell marker expression than the immunogen, although the latter was more effective at inducing antibody production. The chemical carcinogen benzo-α-pyrene at low concentration acted potentially like a mitogen but almost zero immunity was apparent at a carcinogenic dose, with a low profile for LDLR expression and intracellular cholesterol. Conclusion: The findings in our study demonstrate an impact of concentration of BaP on generation of humoral immunity. Probably by immunosuppression through restriction of B-cell populations and associated antibodies, benzo-α-pyrene may exerts carcinogenicity. The level of cholesterol was found to be a pivotal target.

Keywords: B cells; IgG; LDL receptor; cholesterol; tetanus toxoid; pokeweed mitogen; benzo; alpha; pyrene.

Figure 1

Relative expressions of B cells by TT, PWM and BaP. Panel-A, Representation of Flow Cytometry for CD19 and CD79a (B cell markers) labeled cells; Panel-B, Top, Bar graphs showing relative potentiality of immunogen, mitogen and carcinogen on the expression of B-cells evaluated with cell specific combination markers CD19 + CD79a; Bottom, Shows comparison on different treated groups; Respective p-values between the pairs are shown on the top of the bars.

Figure 2

Serum IgG levels by TT, PWM and BaP. Panel-A, Top, Bar graphs showing relative response of immunogen, mitogen and carcinogen on serum IgG levels; Bottom, Graphs showing comparison on different treated groups; Panel-B, Comparison of antibody titre by respective concentrations of TT, PWM, Bap25, Bap-100 showing their relative immunogenic efficiency; Respective p-values between the pairs are shown on the top of the bars.

Figure 3

Relative Expressions of LDLR on CD19 labeled B-Cells by TT, PWM and BaP. Panel-A, Representation of Flow Cytometry for LDLR expression by CD19 B-cells; Panel-B, Top, Bar graphs showing relative potentiality of immunogen, mitogen and carcinogen on the LDLR expression by CD19 B-cells. Mitogens showed dominance over immunogenic and carcinogenic agents; Bottom, Shows comparison of LDLR expression in different treated groups; Respective p-values between the pairs are shown on the top of the bars.

Figure 4

Relative expressions of LDLR on CD79a labeled B-cells by TT, PWM and BaP; Panel-A, Representation of Flow Cytometry for LDLR expression by CD79a B-cells; Panel-B, Top, Bar graphs showing relative potentiality of immunogen, mitogen and carcinogen on the LDLR expression by CD79a B-cells. Mitogens showed dominance over immunogenic and carcinogenic agents; Bottom, Shows comparison of LDLR expression in different treated groups.

Figure 5

Total Cholesterol Concentration in Cytoplasmic and Nuclear Compartments of Lymphocytes. Panel-A, Top, Shows accumulation of more cholesterol in cell cytoplasm and nucleus by typical mitogens (PWM and Bap-25) as compared to immunogen (TT) or carcinogen (Bap-100); Bottom, Shows comparison of cholesterol concentration among different treated groups; Panel-B, Western Blot on LDLR expression of total splenocytes. TT, PWM and BaP25 have shown comparable effect on LDLR expression while carcinogen (BaP100) lowers the cholesterol concentration in normal lymphocytes. Β-actin was used as housekeeping protein marker. Respective p-values between the pairs are shown on the top of the bars.

Figure 6

Spleens from Normal and BaP100 Treated Mice. The histogram shows no apparent structural deformity related to the toxicity of carcinogenic dose, BaP100.