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Abstract
Objective
Interleukin (IL)-17 is a proinflammatory cytokine that has pleiotropic effects on multiple target cells and thereby contributes to the development of immunemediated inflammatory disorders. However, the role of IL-17 in the humoral immune response has not been clearly elucidated.
Methods
Mice deficient in IL-17A (IL-17A knockout [KO] mice) and wild type (WT) C57BL/6 mice were compared. Distinct B cell (mature/precursor and marginal zone/follicular) and plasma cell populations were compared using fluorescence-activated cell sorting (FACS) and confocal immunostaining. Immunoglobulin production was assessed by enzyme-linked immunosorbent assay.
Results
There was no difference in B cell and plasma cell populations between IL-17A KO and WT mice. However, after T cell-dependent antigen challenge, IL-17A KO mice produced lower levels of immunoglobulin (Ig)G1 than wild-type animals. IL-17A KO mice also showed reduced germinal center (GC) formation and lower expression of activation-induced cytidine deaminase, the essential enzyme for class switch recombination (CSR). IL-17 had no effect on the proliferation or survival of naïve B cells in in vitro functional studies. However, IL-17 treatment promoted naïve B cell differentiation into plasma cells in synergy with IL-4, although IL-17 alone had no effect.
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Figure 1.
Comparison of the B cell populations of wild type (WT) and interleukin (IL)-17 knockout (KO) mice. Various B cell subpopulations were compared using FACS analysis. (A) Amongst the B220+ B cells, the subpopulation of precursor B cells (IgD− IgM−), mature B cells (IgD+ IgM−) (left), plasma B cells (CD138+) (middle), follicular B (FOB) cells (CD21 low CD23+), and marginal zone B (MZB) cells (CD21+ CD23 low) (right) were compared. The top panel shows WT and the bottom panel shows IL-17 KO. (B) The number of cells measured by FACS. Data are presented as mean±standard error of the mean. Ig: immunoglobulin, SSC: side scatter.
Figure 2.
Interleukin (IL)-17 contributes to immunoglobulin (Ig) G1 production in mice. Plasma samples (A) and spleen cells (B) were obtained on day 14-post immunization. (A) The levels of anti-4-hydroxy-3-nitrophenacetyl (NP) immunoglobulin in the plasma were measured by enzyme-linked immunosorbent assay (immunostaining, ×400). (B) The levels of total IgG1 positive splenic B cells were measured by FACS analysis. Data are presented as mean±standard error of the mean. WT: wild type, KO: knockout, AID: activation-induced cytidine deaminase. *p-value<0.05.
Figure 3.
Reduced germinal center formation in interleukin (IL)-17 knockout (KO) mice. Spleen tissue was obtained 14 days post immunization. (A) The number of germinal centers was observed on a cross section field using confocal microscopy (immunostaining, ×400). (B) Immunostaining for immunoglobulin (Ig)G1, CD4, and GL-7 was performed. Data are presented as mean±standard error of the mean. WT: wild type, NP: 4-hydroxy-3-nitrophenacetyl. *p-value<0.05.
Figure 4.
The expression of activation-induced cytidine deaminase (AID) is decreased in the splenocytes of interleukin (IL)-17 knockout (KO) mice. The spleens (A) and splenocytes (B) were obtained on day 14-post immunization. (A) Immunostaining for AID, CD4, and GL-7 was performed. (B) The expression of AID in splenocytes was determined by reverse transcription-polymerase chain reaction. Data are presented as mean±standard error of the mean. Representative data are presented. WT: wild type, NP: 4-hydroxy-3-nitrophenacetyl.
Figure 5.
Interleukin (IL)-17 enhances plasma cell differentiation in synergy with IL-4. (A) Spleens were obtained on day 14 after immunization. Immunostaining for CD138, CD4, and GL-7 was performed. (B) Resting B cells were isolated from untreated wild type (WT) mice and cultured with anti-immunoglobulin (Ig)M (5 μ g/mL) in the presence or absence of IL-4 (5 ng/mL) and/or IL-17 (10 ng/mL) for 5 days. Cells were harvested and the frequency of CD138+ B220− cells was measured using FACS. Data are presented as mean±standard error of the mean. NP: 4-hydroxy-3-nitrophenacetyl, KO: knockout.
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